Bright, color-tunable fluorescent dyes in the Vis/NIR region: Establishment of new "tailor-made" multicolor fluorophores based on borondipyrromethene

Article abstract of DOI:10.1002/chem.200801906

A new series of high-performance fluorophores named Keio Fluors (KFL), which are based on borondipyrromethene (BODIPY), are reported. The KFL dyes cover a wide spectral range from the yellow (547 nm) to the near-infrared (NIR, 738 nm) region, and their emission wavelength could be easily and subtly controlled based on simple molecular modifications only, without losing their optical properties. This "tailor-made" synthetic strategy for tuning the emission wavelength enabled the creation of fourteen KFL dyes with well-controlled emission colors (yellow, orange, red, far-red, and NIR). Moreover, these KFL dyes also retain their excellent optical properties, such as spectral bands sharper than quantum dots, high extinction coefficients (140000-316000 M^-1 cm^-1), and high quantum yields (0.56-0.98), without any critical solvent polarity dependent decrease of their brightness. These advantageous characteristics make the KFL dyes potentially useful as new candidates of fluorescent standard dyes to substitute or to complement existing long-wavelength fluorescent dyes, such as cyanines, oxazines, rhodamines, or other BODIPY dyes.

Full text of DOI:10.1002/chem.200801906

DOI: 10.1002/chem.200801906
Bright, Color-Tunable Fluorescent Dyes in the Vis/NIR Region:
Establishment of New “Tailor-Made” Multicolor Fluorophores Based on
Borondipyrromethene
Keitaro Umezawa, Akihiro Matsui, Yuki Nakamura, Daniel Citterio, and Koji Suzuki*[a]
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Chem. Eur. J. 2009, 15, 1096 – 1106

FULL PAPER
Abstract: A new series of high-perfor-
mance fluorophores named Keio
Fluors (KFL), which are based on bor-
ondipyrromethene (BODIPY), are re-
ported. The KFL dyes cover a wide
spectral range from the yellow
(547 nm) to the near-infrared (NIR,
738 nm) region, and their emission
wavelength could be easily and subtly
controlled based on simple molecular
modifications only, without losing their
optical properties. This “tailor-made”
synthetic strategy for tuning the emis-
sion wavelength enabled the creation
of fourteen KFL dyes with well-con-
trolled emission colors (yellow, orange,
red, far-red, and NIR). Moreover,
these KFL dyes also retain their excel-
lent optical properties, such as spectral
bands sharper than quantum dots, high
extinction
coefficients
(140000–
316000mꢀ1 cmꢀ1), and high quantum
yields (0.56–0.98), without any critical
solvent polarity dependent decrease of
their brightness. These advantageous
characteristics make the KFL dyes po-
tentially useful as new candidates of
fluorescent standard dyes to substitute
or to complement existing long-wave-
length fluorescent dyes, such as cya-
nines, oxazines, rhodamines, or other
BODIPY dyes.
Keywords: boron · dyes/pigments ·
fluorescent probes · imaging agents
Introduction
only few dyes which have satisfyingly high brightness (eꢁ
f>105 mꢀ1 cmꢀ1).[5] For instance, some cyanine dyes suffer
from low fluorescence quantum yields (<0.3 in most cases),
rendering their brightness lower than 105 mꢀ1 cmꢀ1.[12]
Bright and long-wavelength emitting fluorescent dyes with
large color variation over a wide spectral region from the
visible (Vis) to the near-infrared (NIR), are of interest in
many fields such as optical engineering, analytical chemistry,
biological chemistry, photochemistry, and others. For exam-
ple, the development of multicolor fluorescent dye standards
is expected to significantly contribute to the progress of fun-
damental and practical technologies: for example, dye laser
series,[1] library of fluorescent reference dyes for photophysi-
cal measurements,[2] and fluorescent probes for multicolor
bioanalysis and bioimaging enabling simultaneous analysis
of multiple analytes.[3–5] Additionally, NIR fluorescent dyes
have been often spotlighted as new revolutionary tools for
noninvasive and simple in vivo optical imaging,[6–10] owing to
the advantages of NIR light between 650–900 nm (often
called “optical window”): for example, significant reduction
of the background signal due to the lowest autoabsorption
and autofluorescence of biomolecules in the NIR region,
low-light scattering and deep penetration of NIR light, and
the possibility to use low-cost excitation light sources.[11]
Ideal multicolor long-wavelength fluorescent dyes should
feature the following characteristics: 1) high fluorescence
quantum yields (f near 1.0); 2) sharp fluorescence spectra
(full width at half maximum height: Dl1/2 around 500 cmꢀ1);
3) large molar extinction coefficients (e over 105 mꢀ1 cmꢀ1);
and 4) ease of tuning the fluorescence emission over a wide
spectral range (Vis/NIR). The brightness, determined by the
product of the extinction coefficient and the quantum yield
(eꢁf), is also an important parameter of each fluorescent
dye. According to a recently published review article, in
which most major fluorophores are introduced, there are
Borondipyrromethene (BODIPY) fluorescent dyes have
recently been focused on due to their intense fluorescence
quantum yields (near 1.0), their sharp absorption and emis-
sion spectra, and their high photostability.[13–17] However,
typical BODIPYs have some drawbacks, such as relatively
short fluorescence emission maxima (lmax around 500 nm)
and low extinction coefficients (e around 80000mꢀ1 cmꢀ1).
Different research groups have used various approaches to
overcome these disadvantages: 1) introduction of electron-
donating substituents,[18,19] 2) replacement of a carbon atom
by a nitrogen atom,[20–22] 3) rigidification of rotatable moiet-
ies,[23–25] 4) extension of the p-conjugation of the struc-
ture,[26,27] and 5) fusion of benzene moieties into the
BODIPY chromophore.[28–30] However, a significant increase
of the extinction coefficients has not been achieved. Instead,
it has been reported, that some modifications resulted in an
undesired decrease of the fluorescence quantum yields and
a hypsochromic shift of the fluorescence maxima.[18,24–26] For
example, the introduction of aryl moieties (phenyl, meth-
ACHUTNGERNoNUG xyAHCTUNGTRNENpUGN henyl, and others) at the a-position of some BODIPYs
has been shown to induce a bathochromic shift, but the opti-
cal properties except for the emission wavelength are gener-
ally not significantly improved. Moreover, the introduction
of ortho-methoxyphenyl groups sometimes invokes more
negative effects, such as a hypsochromic shift, a broadening
of the emission spectra, and a decrease of the extinction co-
efficients and quantum yields.[18,24] These ambiguous correla-
tions between chemical modification to BODIPYs and the
corresponding change of their optical properties make it dif-
ficult to design the desired multicolor fluorescent dyes with-
out losing the advantageous optical properties.
To overcome these drawbacks, we have recently reported
four novel types of heteroaryl-fused BODIPYs, named Keio
Fluors (KFL), with an introduction site for electron-donat-
ing and/or accepting groups.[31] KFL dyes have advantageous
characteristics, such as sharp emission spectra, and high
quantum yields and molar extinction coefficients over a
[a] Dr. K. Umezawa, A. Matsui, Y. Nakamura, Prof. Dr. D. Citterio,
Prof. Dr. K. Suzuki
Department of Applied Chemistry
Faculty of Science and Technology
Keio University, 3-14-1, Hiyoshi
Kohoku-ku, Yokohama 223-8522 (Japan)
Fax : (+81)45-566-1568
E-mail: suzuki@applc.keio.ac.jp
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K. Suzuki et al.
wide spectral range from the visible into the NIR region
(583–738 nm). So far, four basic types of these new dyes
have been introduced. To make full use of the features of
the KFL fluorophores, we extended our investigation to fur-
ther derivatives. Ideal multicolor fluorescent dyes should
allow the easy and fine control of the emission wavelength
over a wide spectral range, with retention of the optical
properties. Preferably, it should also be possible to estimate
the emission wavelength from the chemical modification ap-
plied to the KFL core. In this paper, we report on new strat-
egies enabling the easy control of the emission wavelength:
introduction of several types of electron-donating moieties
(KFL-5–10) and synthesis of asymmetric KFL dyes (KFL-
11–14, shown here).
lished. Asymmetric KFLs were easily synthesized by using
two different pyrroles or furopyrroles according to the same
synthetic scheme.
Spectral properties of phenyl-substituted KFLs (KFL-5–8):
In our previously reported work, the introduction of phenyl
rings into the dyes invoked a drastic bathochromic shift in
the fluorophores with retention of quantum yield and spec-
tral sharpness, and with an increase of the extinction coeffi-
cient. It could also be confirmed by X-ray single-crystal
analysis that almost no torsion between the KFL core and
the phenyl rings occurred, resulting in the increase of the ex-
tinction coefficients.[31] We therefore assumed that this
planar conformation might be crucial for the observed opti-
cal properties. On the basis of this hypothesis, new KFL
dyes (KFL-5–8), which have several types of substituted
phenyl rings, were synthesized and their optical properties
were measured. Specifically, some bulky substituents, such
as methoxy (KFL-6) and isopropyl groups (KFL-8), were in-
troduced in the ortho-position of the phenyl ring to evaluate
the effect of substitution on the conformation and the spec-
tral properties of the fluorophores.
Results and Discussion
Synthesis: The synthetic scheme is outlined in Scheme 1.
Aryl substitutions were performed in the first step by
Suzuki–Miyaura coupling with high yields. Furopyrrole 5-
carboxylic acid segments were synthesized by Hemetsberg-
er–Knittel synthesis, and a-formylation of furopyrroles was
performed in the presence of triethyl orthoformate and tri-
fluoroacetic acid. KFL dyes were obtained from a-formylat-
ed furopyrrole and a-carboxylated furopyrrole in the pres-
ence of trifluoroacetic acid and trichlorophosphate. In this
way, a simple and efficient synthetic scheme could be estab-
As shown in Table 1 and Figure 1, KFL-5–7 exhibited
sharp spectra (Dl1/2 around 500 cmꢀ1), high extinction coeffi-
cients (313000–316000mꢀ1 cmꢀ1) and high quantum yields in
chloroform (0.90–0.97) in the far-red and NIR region
(around 650 nm), and their brightness was in the range of
282000–286000mꢀ1 cmꢀ1. To the best of our knowledge,
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Fluorescent Dyes
FULL PAPER
Scheme 1. Synthetic scheme for Keio Fluors.
Table 1. Optical properties of TM-BDP and Keio Fluors in chloroform.
[a]
labs
lflu
Dl1/2
e[b]
[mꢀ1 cmꢀ1
f[b,c] Brightness[d]
[mꢀ1 cmꢀ1
[nm] [nm] [nm]
G
]
N
]
TM-BDP[31] 509
517
583
620
683
738
661
680
671
655
689
701
549
620
605
644
19 (704)
80000
0.92
73600
KFL-1[31]
KFL-2[31]
KFL-3[31]
KFL-4[31]
KFL-5
579
613
673
723
652
671
662
629
679
690
542
614
596
634
16 (457) 202000
22 (574) 185000
24 (516) 288000
31 (577) 253000
18 (405) 314000
19 (421) 313000
19 (425) 316000
30 (700) 142000
28 (586) 280000
29 (583) 282000
16 (543) 140000
16 (416) 248000
23 (619) 184000
24 (568) 192000
0.96 194000
0.98 181000
0.86 248000
0.56 142000
0.90 286000
0.91 282000
0.97 284000
0.87 118000
0.82 230000
0.81 226000
0.96 134000
0.95 236000
0.95 175000
0.87 167000
KFL-6
KFL-7
KFL-8
KFL-9
KFL-10
KFL-11
KFL-12
KFL-13
KFL-14
[a] The values converted to wavenumbers (cmꢀ1) are shown in parenthe-
ses. [b] Error: within 5%. [c] Reference dyes are listed in the Experimen-
tal Section. [d] Brightness is defined as eꢁf.
there are probably no fluorescent dyes with extinction coef-
ficients over 300000mꢀ1 cmꢀ1 and quantum yields over 0.90
in this far-red region of the spectrum. Their brightness
values (over 200000mꢀ1 cmꢀ1) are higher than those of any
commonly used fluorescent dyes listed in a recent review ar-
ticle.[5] Interestingly, only KFL-8 (ortho-isopropylphenyl-
substituted KFL) showed different spectral characteristics
compared to KFL-3 and KFL-5–7. Dye KFL-8 exhibited a
high quantum yield (0.87), broad spectral band, and low ex-
tinction coefficient at a shorter wavelength (labs: 629 nm)
compared to KFL-5 (phenyl-substituted KFL). The ortho-
methoxyphenyl-substituted KFL-6 on the other hand, exhib-
ited similar characteristics to KFL-3, KFL-5, and KFL-7.
These results demonstrate a certain substituent effect of
ortho-substituted phenyl rings on the spectral properties.
Similar observations have also been reported previously, dis-
Figure 1. a) Normalized absorption and b) fluorescence spectra of (from
left to right) KFL-3 and KFL-5–8 in chloroform.
cussing the effect of ortho-substituents of phenyl rings on
the optical characteristics of BODIPY derivatives.[18,24] Ac-
cording to these reports, the introduction of ortho-methoxy-
phenyl rings into BODIPY or aza-BODIPY at the a-posi-
tions of pyrroles tends to negatively influence their optical
extinction coefficients and quantum yields, shorten the
wavelengths of their absorption and emission maxima, and
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K. Suzuki et al.
broaden their spectral bands. It is stated that this is due to
the twisted conformation between the ortho-methoxyphenyl
rings and the BODIPY core arising from steric hindrance
induced by the ortho-methoxy groups.[24] The twisted form
may allow flexible rotation of the phenyl rings and hence,
have an effect on the extinction coefficients, quantum yields
and spectral sharpness. Therefore, the twisting angle be-
tween the phenyl rings and the BODIPY core is considered
to relate to the trend of the optical properties.
In the case of KFL-4, a planar conformation between the
KFL core and the phenyl rings was confirmed by X-ray
single-crystal analysis. Also in the case of KFL-3 and KFL-
6, ring coplanarity is assumed based on the results of AM1
quantum calculation (Figure 2). For KFL-6, this is further
supported by NMR measurements. As shown in Figure 3,
the chemical shifts of the Hc protons (3- and 7-positions of
the KFL core) are most strongly influenced by the substitu-
tion pattern of the phenyl rings. Especially in the case of
KFL-6, the chemical shift of Hc shifted downfield (d=
7.3 ppm) compared to the other KFL-3, KFL-5, KFL-7, and
KFL-8 (d=6.7–6.9 ppm). This is assumed to be due to the
presence of the O atoms (at the ortho position of the phenyl
rings) near the Hc protons, indicating a certain interaction
through space.
In contrast, AM1 calculation for KFL-8 indicated an
almost orthogonal orientation between the phenyl rings and
the KFL core, owing to the presence of the bulky isopropyl
units at the ortho position of the phenyl rings (Figure 2).
This resulted in a decrease of the extinction coefficient, a
hypsochromic shift of the spectral maxima, and reduced
spectral sharpness. The quantum yield remains almost unaf-
fected (0.87), implying that the phenyl rings are hindered in
rotation by the bulky isopropyl units. Previous research into
BODIPY derivatives (with phenyl rings at the a-position
forced into an orthogonal orientation by bulky methyl
groups) showed quite similar results,[32] which further sup-
ports our considerations. Consequently, it is suggested that
coplanarity of the KFL core and the phenyl substituents is a
prerequisite for the desired advantageous spectral proper-
ties, and that a twisted conformation results in a reduction
of certain properties.
Spectral properties of dimethoxyphenyl-substituted KFL
dyes (KFL-9 and KFL-10): As discussed above, we discov-
ered a positive effect of ortho-methoxy substitution of
phenyl rings on the optical properties of the KFL fluoro-
phores (red-shift of absorption and fluorescence emission
with sharp spectra, high extinction coefficient and fluores-
cence quantum yield), although it had been reported to
evoke a negative effect in the case of BODIPYs. It also im-
plies that further spectral shift can be expected by increasing
the number of electron donors at the phenyl ring. On the
basis of this consideration, dimethoxyphenyl-substituted
KFL dyes (KFL-9 and KFL-10) were designed, synthesized,
and characterized regarding the degree of spectral shift.
As shown in Figure 4, KFL-9 and KFL-10 exhibited signif-
icantly long-wavelength-shifted fluorescence reaching up to
700 nm, while retaining high extinction coefficients
(ꢁ282000mꢀ1 cmꢀ1), high quantum yields (ꢁ0.82), and sharp
spectra relative to KFL-5 (benzene-substituted KFL) in
chloroform. These results are noteworthy, because the previ-
ously reported dimethoxyphenyl-substituted BODIPY dyes
exhibited less positive properties: broader spectra, low ex-
tinction coefficient, and relatively short or identical wave-
length.[24] In the case of KFL, fine tuning of absorption and
fluorescence emission peak maxima could be achieved by
simply attaching the methoxy units at various positions of
the phenyl rings.
Figure 2. a) X-ray molecular structure of KFL-4[31] and AM1 calculated
molecular structures of b) KFL-5, c) KFL-6, and d) KFL-8. Top figures
and bottom figures in each dye represent front view and side view, re-
spectively.
Spectral properties of asymmetric KFL dyes (KFL-11–14):
Asymmetric KFL dyes were expected to expand the fluores-
cence wavelength variations. Therefore, four asymmetric
dyes (KFL-11–14) were designed and synthesized. For ex-
Figure 3. NMR spectra of KFL-3 and KFL-5–8 in [D1]chloroform at
room temperature.
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Fluorescent Dyes
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Figure 4. a) Normalized absorption and b) fluorescence spectra of (from
left to right) KFL-3, KFL-5, KFL-6, KFL-9, and KFL-10 in chloroform.
Figure 5. a) Normalized absorption and b) fluorescence spectra of (from
left to right) TM-BDP, KFL-11, KFL-1, KFL-12, and KFL-5 in chloro-
form.
ample, asymmetric KFL-11, which is a combination of the
1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-inda-
cene (TM-BDP) and the KFL-1 backbone, was expected to
exhibit its absorption/fluorescence peak maxima centered
between those of TM-BDP and KFL-1 (probably at around
540/550 nm). The experimental result showed that KFL-11
exhibited a vivid pink absorption color (542 nm) and intense
yellow emission color (549 nm, f=0.96), very close to the
expected peak wavelengths (Figures 5 and 6). Based on
these results, several types of asymmetric KFL (KFL-12–14)
were synthesized to obtain new fluorescent dyes the emis-
sion peaks of which can be flexibly controlled. The dye
KFL-12, which is a combination of KFL-1 and KFL-5, also
exhibited its absorption/emission peak at 614/620 nm, which
is very close to the arithmetic mean of the combination of
KFL-1 (579/583 nm) and KFL-5 (652/661 nm). The absorp-
tion and fluorescence maxima of KFL-2 and KFL-12 are
almost identical, with the spectral broadness and the extinc-
tion coefficient of KFL-12 being improved. The absorption/
emission maxima of KFL-13 and KFL-14, which have one
2,4-dimethoxyphenyl moiety, are also centered between the
corresponding values of TM-BDP/KFL-10, and KFL-1/KFL-
10, respectively. The absorption maxima of KFL-11 and
KFL-14 are just identical to the excitation wavelengths of
the green He–Ne laser (543 nm) and the red He–Ne laser
(633 nm), respectively, which are widely used as standard
lasers for confocal laser fluorescence microscopy and flow
cytometry. This indicates that very efficient excitation of
Figure 6. Absorption (upper row) and fluorescence (bottom row) colors
of TM-BDP and KFLs in chloroform.
KFL-11 and KFL-14 using existing standard lasers could be
realized. Therefore, with asymmetric KFL dyes, a fine-
tuning of absorption/emission wavelengths to a desired
region could be achieved relying on a very simple combina-
tion of KFL dyes without losing their optical properties.
Effect of solvent polarity: The effect of solvent polarity on
the optical properties of the KFL dyes was investigated, be-
cause many fluorescent dyes are influenced by the solvent
polarity, and their optical characteristics (especially quantum
yields) are changed. All KFL dyes exhibited small blue
shifts (about 10 nm) with increasing solvent polarity, as typi-
cal BODIPY dyes exhibit the same trend.[33]
In terms of quantum yields, KFL-1 and KFL-5 showed
almost no fluorescence decrease, and retained extremely
high quantum yields even in polar solvents, such as ethanol
and methanol (Table 2). KFL-3 and KFL-10 also exhibited
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K. Suzuki et al.
Table 2. Fluorescence quantum yields of selected KFLs in various sol-
vents.[a]
Table 3. Optical properties of typical long-wavelength fluorescent dyes.
labs/lflu
e
f
Brightness
[mꢀ1 cmꢀ1
Toluene
CHCl3
THF
MeCN
EtOH
MeOH
[nm]
[mꢀ1 cmꢀ1
]
U
]
KFL-1
KFL-2
KFL-3
KFL-4
KFL-5
KFL-10
KFL-12
0.94
0.97
0.87
0.58
0.89
0.83
0.98
0.96
0.98
0.86
0.56
0.90
0.81
0.96
0.93
0.92
0.83
0.49
0.89
0.75
0.97
0.95
0.84
0.85
0.45
0.88
0.76
0.95
0.97
0.77
0.77
0.37
0.86
0.68
0.93
0.96
0.70
0.77
0.32
0.87
0.63
0.96
rhodamine dyes
R6G[34]
530/556
540/565
575/590
116000[37]
95000
0.95[a]
0.68[b]
0.95[a]
110200
64600
132000
TMR[38]
ShR101[39]
139000[37]
oxazine dyes
cresyl violet[35]
nile blue[40]
BODIPY dyes
restricted BODIPY[25]
aza-BODIPY[20]
restricted aza-BODIPY[23]
cyanine dyes[41]
Cy3B
598/620
625/649
83000[37]
76800[37]
0.54[b]
0.27[a]
44800
20700
[a] Reference dyes are listed in the Experimental Section.
619/629
688/714
740/751
145750
83000
159000
0.72[c]
0.36[c]
0.28[c]
105000
29900
44500
quantum yields high enough for bioimaging applications, al-
though a slight fluorescence decrease was observed with in-
creasing solvent polarity. This slight fluorescence decrease
of KFL-10 in polar solvents is assumed to be due to the
presence of the electron-donating moieties, which cause in-
tramolecular charge transfer (ICT). ICT is known to influ-
ence the rate of nonradiative relaxation of fluorophores, re-
sulting in modified fluorescence quantum yields. Similar ten-
dencies are found in the case of KFL-2 and KFL-4, which
have an electron-accepting moiety (-CF3). On the other
hand, KFL-12, which has almost the same absorption/emis-
sion maxima as KFL-2, but lacks electron-donating/accept-
ing moieties, showed no fluorescence quenching and exhibit-
558/572
649/670
675/694
743/767
130000
250000
250000
200000
0.67[d]
0.28[d]
0.28[d]
0.28[d]
87100
70000
70000
56000
Cy5
Cy5.5
Cy7
[a] In ethanol. [b] In methanol. [c] In chloroform. [d] In aqueous buffer.
brightness of KFL-1 and KFL-11 is still higher than that of
rhodamines. Also, the optical properties of KFL-2, KFL-5,
and KFL-12–14 are generally superior to those of oxazines
(e.g., Cresyl violet, Nile blue), which are widely used as red
fluorescent dye standards. The emission maxima of KFL-7,
KFL-9, and KFL-4 are almost identical to Cy5, Cy5.5, and
Cy7, respectively, but the brightness of these three KFL
dyes is higher than those of cyanine dyes. Considering long-
wavelength emitting BODIPYs, the dyes of the KFL series
are among the most high-performance BODIPY derivatives.
In terms of spectral sharpness, the spectral bands of KFL
dyes are sharper than those of almost all existing fluorescent
dyes. Moreover, KFL dyes exhibit sharper emission spectra
than quantum dots, which are known as fluorescent materi-
ed high fluorescence in any solvent (fEtOH =0.93, fMeOH
=
0.96). Thus, a significant improvement in terms of fluores-
cence quantum yields of red fluorescent dyes emitting
around 620 nm could be achieved. These high quantum
yields in any solvent are promising for high-sensitive and
high-resolution analysis even in aqueous solution, although
the KFL dyes in their present state are not sufficiently solu-
ble in water. To evaluate the photostability of KFLs in aque-
ous solution (DMSO/water=50:50 and 70:30 for KFL-1 and
KFL-5, respectively), the decrease of the fluorescence inten-
sity of KFL-1 and KFL-5 was monitored upon continuous ir-
radiation with white light from a Xe lamp (150 W, without
passing the monochromator) at 258C for one hour, after
which over 96% of fluorescence intensity was retained in
both cases.
als with sharp emission spectra (e.g., Qdot 605: lflu
=
655 nm, Dl1/2 =26 nm in methanol, Qdot 705: lflu =705 nm,
Dl1/2 =74 nm in methanol). These sharp spectra enable easy
spectral separation, and therefore, allow high-resolution
multicolor bioanalysis and bioimaging.
Conclusion
Comparison of KFL spectral properties to commercially
available fluorescent dyes: Optical properties of some com-
mercially available long-wavelength emitting dyes (e.g.,
rhodamine, oxazine, BODIPY, cyanine) are summarized in
Table 3. It can be concluded that the KFL dyes are at least
equivalent and often superior to most of the commercially
available fluorescent dyes in terms of extinction coefficient,
quantum yield, spectral sharpness, and brightness (Tables 1
and 3). The most remarkable fact is that KFL dyes exhibit
extremely high brightness (118000–286000mꢀ1 cmꢀ1), since
only few fluorescent dyes with brightness values over
100000mꢀ1 cmꢀ1 are reported.[5] Rhodamine dyes and Cy3B,
which are among the most widely used yellow-red fluores-
cent dye standards, show high optical properties with bright-
ness around 100000mꢀ1 cmꢀ1. The emission maxima of KFL-
1 and KFL-11 are identical to some rhodamine dyes, but the
In summary, we could establish a new series of long-wave-
length fluorescent dyes, named Keio Fluors (KFLs), based
on BODIPY with sophisticated optical properties, such as
vivid colors in the Vis/NIR region (yellow, orange, red, far-
red, and NIR), high extinction coefficients (140000–
316000mꢀ1 cmꢀ1), high quantum yields (0.56–0.98), high
brightness (118000–286000mꢀ1 cmꢀ1), and emission bands
even sharper than quantum dots. In addition, chemical
modification of the KFL dyes allows the easy and fine
tuning of absorption/emission peaks without negatively in-
fluencing their optical properties. Simple and accurate esti-
mation of the absorption/emission wavelength is possible
based on the substituents attached to the BODIPY cores:
for example, fusion of one furan part (ca. +30 nm), intro-
duction of one phenyl ring (ca. +40 nm), and attachment of
1102
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Chem. Eur. J. 2009, 15, 1096 – 1106

Fluorescent Dyes
FULL PAPER
was obtained as a yellow liquid (915 mg, 73.8%). Eluent for chromatog-
one methoxy moiety at ortho- or para-position of a phenyl
ring (ca. +10 nm). Therefore, various tailor-made fluores-
cent dyes with desired absorption/emission wavelength
could be easily designed and synthesized solely relying on
the simple estimation of these factors. To the best of our
knowledge, KFL dyes show higher optical performance than
any other known fluorescent dyes in the long-wavelength
region (around 550–750 nm). These properties make the
KFL series dyes very promising for new fluorescent stan-
dard dyes.
1
raphy: n-hexane/chloroform: 80:20!60:40. H NMR (CDCl3): d=9.67 (s,
1H), 7.57 (d, J=6.9 Hz, 1H), 7.44–7.41 (m, 2H), 7.34 (d, J=3.9 Hz, 1H),
7.29–7.24 (m, 1H), 6.66 (d, J=3.6 Hz, 1H), 3.44–4.40 (m, 1H), 1.29 (s,
3H), 1.26 ppm (s, 3H).
5-(2,5-Dimethoxyphenyl)-furan-2-carbaldehyde (2g): 2,5-Dimethoxyphe-
nylboronic acid (3.64 g, 20.0 mmol) was used as the starting material, and
2g was obtained as a yellow liquid (3.82 g, 82.2%). Eluent for chroma-
tography: n-hexane/chloroform 50:50!20:80. 1H NMR (CDCl3): d=9.65
(s, 1H), 7.56 (t, J=1.7 Hz, 1H), 7.33 (d, J=3.7 Hz, 1H), 7.17 (d, J=
3.7 Hz, 1H), 6.93 (d, J=1.7 Hz, 2H), 6.72 (d, J=3.6 Hz, 1H), 3.86 ppm
(s, 3H).
5-(2,4-Dimethoxyphenyl)-furan-2-carbaldehyde (2h): 2,4-Dimethoxyphe-
nylboronic acid (2.50 g, 13.7 mmol) was used as the starting material, and
2h was obtained as a yellow liquid (2.47 g, 78.3%). Eluent for chroma-
tography: n-hexane/chloroform 50:50!20:80. 1H NMR (CDCl3): d=9.58
(s, 1H), 7.97 (d, J=8.8 Hz, 1H), 7.31 (d, J=3.6 Hz, 1H), 7.00 (d, J=
3.6 Hz, 1H), 6.60 (d, J=8.8 Hz, 1H), 6.53 (s, 1H), 3.94 (s, 3H), 3.87 ppm
(s, 3H).
Experimental Section
Materials: All chemical reagents and solvents for synthesis were pur-
chased from commercial suppliers (Wako Pure Chemical, Tokyo Kasei
Industry, and Aldrich Chemical), and used without further purification.
All moisture-sensitive reactions were carried out under an atmosphere of
argon. The composition of mixed solvents is given by the volume ratio
(v/v). Synthetic procedures for KFL-1–4 are described in the literature.[31]
Instruments: 1H NMR and 13C NMR spectra were recorded on a JEOL
JNM-LA 300 (JEOL, Tokyo, Japan) or Varian MVX-300 (Varian, Palo
Alto, CA) spectrometer at room temperature (if no specific temperature
is indicated). The measurements were performed at 300 MHz (for 1H)
and 75 MHz (for 13C), respectively. All chemical shifts are relative to an
internal standard of tetramethylsilane (d=0.0 ppm), and coupling con-
stants are given in Hz. Flash chromatography separation was undertaken
General procedure for synthesis of 2-substituted 4H-furoACTHNUTRGNEU[GN 3,2-b]pyrrole-5-
carboxylic acid ethyl esters 3c–3h: 5-Substituted furan-2-carbaldehyde
2c–2h (1.0 equiv) and ethyl azidoacetate (2.0 equiv) were dissolved in an-
hydrous ethanol (100 mL) and stirred at 08C. A solution of sodium eth-
oxide (20 wt% in ethanol, 2.0 equiv) was added dropwise into the mix-
ture, and stirring was continued for 2–4 h until the reaction was over.
Excess saturated aqueous NH4Cl solution was added to form a precipi-
tate, which was collected by filtration. The precipitate was washed with
water and dried in vacuo. In the cases in which no precipitate was
formed, the water phase was extracted with ethyl acetate, and the organic
phase then washed with water and brine, dried over Na2SO4, and evapo-
rated. The resulting residue was dissolved in toluene (30 mL) and heated
to reflux for 1 h. After cooling, the solvent was evaporated. The residue
was purified by recrystallization from n-hexane/ethyl acetate mixture, or
using
a YFLC-Al-560 chromatograph (Yamazen, Osaka, Japan).
MALDI-TOF (matrix-assisted laser desorption ionization—time-of-
flight) mass spectra were recorded on an Ultraflex TOF/TOF spectrome-
ter (Bruker) with a-cyano-4-hydroxycinnamic acid (CHCA) as matrix.
by chromatography (silica gel) to obtain the 2-substituted 4H-furo
b]pyrrole-5-carboxylic acid ethyl ester.
ACHTUNGTRENNUNG[3,2-
General procedure for the synthesis of 5-substituted furan-2-carbalde-
hydes 2c–2h: Aryl boronic acid (1.0 equiv) and 5-bromo-2-furaldehyde
(1.0 equiv) were dissolved in a mixture of toluene (100 mL), ethanol
(20 mL), and an aqueous solution of Na2CO3 (2m, 20 mL), and degassed
in vacuo. [1,1’-Bis(diphenylphosphino)ferrocene]palladium(II) dichloride
dichloromethane complex (1:1; 0.02 equiv) was added to the solution,
and the mixture was heated at 808C while monitoring the reaction prog-
ress with TLC. After the reaction was completed (30 min–overnight), the
mixture was cooled to room temperature and the organic phase was
washed with water and brine, dried over Na2SO4, and evaporated. The re-
sulting residue was purified by flash chromatography (silica gel) to obtain
the arylfuran-2-carbaldehyde.
2-Phenyl-4H-furoACTHUNRGTNE[NUG 3,2-b]pyrrole-5-carboxylic acid ethyl ester (3c): Com-
pound 2c (2.85 g, 16.6 mmol) was used as the starting material, and 3c
was obtained as a yellow solid (1.77 g, 41.9%). Purification: recrystalliza-
tion. 1H NMR (CDCl3): d=8.69 (s, 1H), 7.73 (d, J=7.8 Hz, 2H), 7.41 (t,
J=7.2 Hz, 2H), 7.29 (t, J=7.5 Hz, 1H), 6.82 (s, 1H), 6.72 (s, 1H), 4.36
(q, J=7.2 Hz, 2H), 1.39 ppm (t, J=7.2 Hz, 3H).
2-(2-Methoxyphenyl)-4H-furoACHTNUTRGNEUNG[3,2-b]pyrrole-5-carboxylic acid ethyl ester
(3d): Compound 2d (2.66 g, 13.2 mmol) was used as the starting material,
and 3d was obtained as a yellow solid (1.53 g, 40.8%). Purification: re-
crystallization. 1H NMR (CDCl3): d=8.69 (brs, 1H), 7.97 (dd, J=7.7,
1.7 Hz, 1H), 7.28 (dt, J=7.8, 1.8 Hz, 1H), 7.05 (t, J=7.5 Hz, 1H), 7.07 (s,
1H), 6.81 (s, 1H), 4.36 (q, J=7.2 Hz, 2H), 3.98 (s, 3H), 1.39 ppm (t, J=
7.2 Hz, 3H).
5-Phenyl-furan-2-carbaldehyde (2c): Phenylboronic acid (2.50 g,
20.5 mmol) was used as the starting material and 2c was obtained as a
yellow liquid (3.15 g, 89.2%). Eluent for chromatography: n-hexane/ethyl
acetate 80:20!50/50. 1H NMR (CDCl3): d=9.65 (s, 1H), 7.83 (dd, J=
8.4, 1.3 Hz, 2H), 7.48–7.39 (m, 3H), 7.32 (d, J=3.9 Hz, 1H), 6.84 ppm (d,
J=3.6 Hz, 1H).
2-(4-Isopropylphenyl)-4H-furoACTHNUGRTENUNG[3,2-b]pyrrole-5-carboxylic acid ethyl ester
(3e): Compound 2e (885 mg, 4.13 mmol) was used as the starting materi-
al, and 3e was obtained as a brown solid (465 mg, 37.9%). Purification:
recrystallization. 1H NMR (CDCl3): d=8.69 (brs, 1H), 7.67 (d, J=
6.6 Hz, 2H), 7.28 (d, J=6.6 Hz, 2H), 6.82 (s, 1H), 6.67 (s, 1H), 4.36 (q,
J=7.2 Hz, 2H), 2.98–2.89 (m, 1H), 1.39 (t, J=7.2 Hz, 3H), 1.29 (s, 3H),
1.27 ppm (s, 3H).
5-(2-Methoxyphenyl)-furan-2-carbaldehyde (2d): 2-Methoxyphenylbor-
onic acid (2.60 g, 17.1 mmol) was used as the starting material, and 2d
was obtained as a yellow liquid (2.86 g, 82.7%). Eluent for chromatogra-
phy: n-hexane/ethyl acetate 80:20!50:50. 1H NMR (CDCl3): d=9.64 (s,
1H), 8.04 (dd, J=7.8, 1.8 Hz, 1H), 7.36 (dt, J=7.8, 1.5 Hz, 1H), 7.33 (d,
J=3.6 Hz, 1H), 7.13 (d, J=3.6 Hz, 1H), 7.06 (dt, J=7.8, 0.6 Hz, 1H),
6.99 (d, J=8.1 Hz, 1H), 3.96 ppm (s, 1H).
2-(2-Isopropylphenyl)-4H-furoACTHNUGRTENUNG[3,2-b]pyrrole-5-carboxylic acid ethyl ester
(3 f): Compound 2 f (900 mg, 4.20 mmol) was used as the starting materi-
al, and 3 f was obtained as a brown solid (390 mg, 31.2%). Purification:
chromatography (eluent: n-hexane/chloroform 50:50!20:80). 1H NMR
(CDCl3): d=8.68 (brs, 1H), 7.53 (d, J=7.1 Hz, 1H), 7.44–7.34 (m, 2H),
7.27–7.22 (m, 1H), 6.84 (dd, J=1.0, 1.7 Hz, 1H), 6.50 (d, J=0.8 Hz, 1H),
4.36 (q, J=7.1 Hz, 2H), 3.48–3.39 (m, 1H), 1.39 (t, J=7.3 Hz, 3H), 1.28
(s, 3H), 1.26 ppm (s, 3H).
5-(4-Isopropylphenyl)-furan-2-carbaldehyde (2e): 4-Isopropylphenylbor-
onic acid (968 mg, 5.90 mmol) was used as the starting material, and 2e
was obtained as a yellow liquid (885 mg, 70.0%). Eluent for chromatog-
raphy: n-hexane/chloroform 50:50!20:80. 1H NMR (CDCl3): d=9.63 (s,
1H), 7.76 (d, J=8.5 Hz, 2H), 7.32 (d, J=3.7 Hz, 1H), 7.31 (d, J=8.3 Hz,
2H), 6.80 (d, J=3.7 Hz, 1H), 3.00–2.90 (m, 1H), 1.29 (s, 3H), 1.27 ppm
(s, 3H).
2-(2,5-Dimethoxyphenyl)-4H-furoACTHNUTRGNE[NUG 3,2-b]pyrrole-5-carboxylic acid ethyl
ester (3g): Compound 2g (3.82 g, 16.4 mmol) was used as the starting
material, and 3g was obtained as a yellow solid (2.70 g, 52.1%). Purifica-
tion: recrystallization. 1H NMR (CDCl3): d=8.71 (brs, 1H), 7.51 (d, J=
5-(2-Isopropylphenyl)-furan-2-carbaldehyde (2 f): 2-Isopropylphenylbor-
onic acid (950 mg, 5.79 mmol) was used as the starting material, and 2 f
Chem. Eur. J. 2009, 15, 1096 – 1106
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1103

K. Suzuki et al.
3.3 Hz, 1H), 7.09 (s, 1H), 6.90 (d, J=9.0 Hz, 1H), 6.84–6.80 (m, 2H),
4.36 (q, J=7.2 Hz, 2H), 3.92 (s, 3H), 3.86 (s, 3H), 1.39 ppm (t, J=7.2 Hz,
3H).
9.32 (brs, 1H), 7.76 (d, J=7.2 Hz, 2H), 7.43 (t, J=7.5 Hz, 2H), 7.34 (t,
J=7.2 Hz, 1H), 6.79 (s, 1H), 6.76 ppm (s, 1H).
2-(2-Methoxyphenyl)-4H-furoACTHNUGRTENUNG[3,2-b]pyrrole-5-carbaldehyde (5d): Com-
2-(2,4-Dimethoxyphenyl)-4H-furoACTHNUTRGNE[NUG 3,2-b]pyrrole-5-carboxylic acid ethyl
pound 4d (50.0 mg, 0.194 mmol) was used as the starting material, and
5d was obtained as a yellow solid (41.0 mg, 87.4%). Eluent for chroma-
tography: n-hexane/ethyl acetate: 75:25!60:40. 1H NMR (CDCl3): d=
9.49 (brs, 1H), 9.44 (s, 1H), (dd, J=7.7, 1.7 Hz, 1H), 7.32 (dt, J=7.5,
1.8 Hz, 1H), 7.11 (s, 1H), 7.06 (dt, J=7.8, 0.9 Hz, 1H), 6.77 (s, 1H),
3.98 ppm (s, 1H).
ester (3h): Compound 2h (2.33 g, 10.0 mmol) was used as the starting
material, and 2h was obtained as a yellow solid (932 mg, 27.0%). Purifi-
1
cation: chromatography (eluent: chloroform/ethyl acetate 95:5). H NMR
(CDCl3): d=8.65 (brs, 1H), 7.87 (d, J=8.5 Hz, 1H), 6.91 (s, 1H), 6.78 (s,
1H), 6.59 (d, J=8.6 Hz, 1H), 6.54 (s, 1H), 4.35 (q, J=7.2 Hz, 2H), 3.94
(s, 3H), 3.86 (s, 3H), 1.38 ppm (t, J=7.1 Hz, 3H).
2-(4-Isopropylphenyl)-4H-furoACTHUNRTGNEUNG[3,2-b]pyrrole-5-carbaldehyde (5e): Com-
General procedure for synthesis of 2-substituted 4H-furoACTHNUTRGNE[NUG 3,2-b]pyrrole-
pound 4e (32.2 mg, 0.120 mmol) was used as the starting material, and
5e was obtained as a gray solid (25.1 mg, 82.8%). Eluent for chromatog-
raphy: chloroform/ethyl acetate 98:2. 1H NMR (CDCl3): d=9.52 (brs,
1H), 9.44 (s, 1H), 7.70 (d, J=8.1 Hz, 2H), 7.29 (d, J=8.4 Hz, 2H), 6.78
(s, 1H), 6.71 (s, 1H), 2.99–2.90 (m, 1H), 1.29 (s, 3H), 1.27 ppm (s, 3H).
5-carboxylic acids 4c–4h: NaOH (ca. 15 equiv) in water (5 mL) was
added to a solution of 2-substituted acid ethyl esters 3c–3h (1.0 equiv) in
ethanol (10 mL) and the mixture was refluxed for 30 min. After cooling,
concentrated aqueous HCl was added until a precipitate formed, which
was filtered. The resulting precipitate was washed with water and dried
2-(2-Isopropylphenyl)-4H-furoACTHUNRTGNEUNG[3,2-b]pyrrole-5-carbaldehyde (5 f): Com-
pound 4 f (27.8 mg, 0.103 mmol) was used as the starting material, and 5 f
in vacuo to obtain the 2-substituted 4H-furo
acid 4c–4h.
ACHTUNGTNER[NUNG 3,2-b]pyrrole-5-carboxylic
was obtained as a gray solid (25.0 mg, 95.8%). Eluent for chromatogra-
1
2-Phenyl-4H-furoACHTUNGTRENNUNG[3,2-b]pyrrole-5-carboxylic acid (4c): Compound 3c
phy: chloroform/ethyl acetate 98:2. H NMR (CDCl3): d=9.88 (brs, 1H),
(1.57 g, 6.15 mmol) was used as the starting material, and 4c was ob-
tained as a gray solid (1.34 g, 95.5%). 1H NMR ([D6]DMSO): d=12.41
(s, 1H), 11.62 (s, 1H), 7.80 (d, J=7.5 Hz, 2H), 7.43 (t, J=7.5 Hz, 2H),
7.31 (t, J=7.8 Hz, 1H), 7.14 (s, 1H), 6.73 ppm (s, 1H).
9.47 (s, 1H), 7.54 (d, J=7.8 Hz, 1H), 7.46–7.37 (m, 2H), 7.26 (t, J=
7.2 Hz, 2H), 6.81 (s, 1H), 6.56 (s, 1H) 3.48–3.39 (m, 1H), 1.29 (s, 3H),
1.27 ppm (s, 3H).
2-(2,5-Dimethoxyphenyl)-4H-furo
G
(5g):
2-(2-Methoxyphenyl)-4H-furoACHTNUTRGNEUNG[3,2-b]pyrrole-5-carboxylic acid (4d): Com-
Compound 4g (36.6 mg, 0.127 mmol) was used as the starting material,
and 5g was obtained as a gray solid (30.2 mg, 87.4%). Eluent for chro-
matography: chloroform/ethyl acetate 95:5. 1H NMR (CDCl3): d=9.57
(brs, 1H), 9.44 (s, 1H), 7.52 (d, J=3.0 Hz, 1H), 7.14 (s, 1H), 6.92 (d, J=
9.3 Hz, 1H), 6.87 (dd, J=9.0 Hz, 3.0 Hz, 1H), 6.77 (s, 1H), 3.93 (s, 1H),
3.86 ppm (s, 1H).
pound 3d (1.38 g, 4.84 mmol) was used as the starting material, and 4d
was obtained as a green solid (1.15 g, 92.4%). 1H NMR ([D6]DMSO):
d=12.41 (s, 1H), 11.56 (s, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.32 (t, J=
7.5 Hz, 1H), 7.15 (d, J=8.4 Hz, 1H), 7.05 (t, J=8.7 Hz, 1H), 7.01 (s,
1H), 6.71 (s, 1H), 3.96 ppm (s, 3H).
2-(4-Isopropylphenyl)-4H-furoACTHNUGRTENUNG[3,2-b]pyrrole-5-carboxylic acid (4e): Com-
2-(2,4-Dimethoxyphenyl)-4H-furo
G
(5h):
pound 3e (345 mg, 1.16 mmol) was used as the starting material, and 4e
was obtained as a gray solid (284 mg, 91.0%). 1H NMR ([D6]DMSO):
d=12.40 (s, 1H), 11.57 (s, 1H), 7.72 (d, J=8.1 Hz, 1H), 7.31 (d, J=
8.4 Hz, 1H), 7.05 (s, 1H), 6.70 (s, 1H), 2.95–2.86 (m, 1H), 1.23 (s, 3H),
1.21 ppm (s, 3H).
Compound 4h (28.1 mg, 0.098 mmol) was used as the starting material,
and 5h was obtained as a gray solid (22.4 mg, 84.9%). Eluent for chro-
matography: chloroform/ethyl acetate 95:5. 1H NMR (CDCl3): d=9.41
(s, 1H), 9.07 (brs, 1H), 7.89 (d, J=8.8 Hz, 1H), 6.95 (s, 1H), 6.74 (s,
1H), 6.61 (dd, J=8.8, 2.2 Hz, 1H), 6.55 (d, J=2.2 Hz, 1H), 3.96 (s, 3H),
3.87 ppm (s, 3H).
2-(2-Isopropylphenyl)-4H-furoACTHNUGRTENUNG[3,2-b]pyrrole-5-carboxylic acid (4 f): Com-
pound 3 f (300 mg, 1.01 mmol) was used as the starting material, and 4 f
was obtained as a gray solid (252 mg, 92.7%). 1H NMR ([D6]DMSO):
d=12.40 (s, 1H), 11.59 (s, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.47 (d, J=
7.8 Hz, 1H), 7.40 (t, J=7.2 Hz, 1H), 7.28 (t, J=7.5 Hz, 1H), 6.74 (s, 1H),
6.66 (s, 1H), 3.43–3.34 (m, 1H), 1.23 (s, 3H), 1.21 ppm (s, 3H).
General procedure for synthesis of symmetric KFL dyes (KFL-5–10):
Compounds 4c–4h (1 equiv) were dissolved in trifluoroacetic acid (1 mL)
and heated at 508C for 10 min. Compounds 5c–5h (1 equiv) and phos-
phoryl chloride (0.8 mL) were added into the reaction mixture, and
stirred for further 10 min until an intense color was formed. After cool-
ing, the reaction mixture was poured into saturated aqueous NaHCO3 so-
lution to neutralize. The formed precipitate was filtered, washed with
water, and dried in vacuo. The resulting compound was dissolved in
1,1,2-trichloroethane (10 mL), before BF3·Et2O (0.3 mL) and triethyl-
2-(2,5-Dimethoxyphenyl)-4H-furoACTHNUTRGNE[NUG 3,2-b]pyrrole-5-carboxylic acid (4g):
Compound 3g (2.67 g, 8.47 mmol) was used as the starting material, and
1
4g was obtained as a green solid (1.90 g, 78.1%). H NMR ([D6]DMSO):
d=12.42 (s, 1H), 11.57 (s, 1H), 7.35 (d, J=3.0 Hz, 1H), 7.08 (d, J=
9.0 Hz, 1H), 7.04 (s, 1H), 6.89 (dd, J=9.3, 3.3 Hz, 1H), 6.72 (s, 1H), 3.91
(s, 3H), 3.78 ppm (s, 3H).
AHCTUNGTREGaNNUN mine (0.2 mL) were added and the mixture was stirred at 1008C for
15 min. After cooling, the reaction mixture was diluted with chloroform
and washed with saturated aqueous NaHCO3 solution and brine, dried
over Na2SO4, and evaporated. The residue was purified by chromatogra-
phy (silica gel) to obtain the KFL dyes (KFL-5–10).
2-(2,4-Dimethoxyphenyl)-4H-furoACTHNUTRGNE[NUG 3,2-b]pyrrole-5-carboxylic acid (4h):
Compound 3h (0.91 g, 2.89 mmol) was used as the starting material, and
4h was obtained as a green solid (0.78 g, 94%). 1H NMR ([D6]DMSO):
d=12.34 (s, 1H), 11.49 (s, 1H), 7.74 (d, J=8.4 Hz, 1H), 6.84 (s, 1H),
6.70–6.68 (m, 2H), 6.66 (dd, J=8.4, 2.1 Hz, 1H), 3.95 (s, 3H), 3.82 ppm
(s, 3H).
2,8-Diphenyl-difuroACTHNUGRTENNUG[2,3-b]ACHTUNGTRNE[NUGN 3,2-g]-5,5-difluoro-5-bora-3a,4a-diaza-s-inda-
cene (KFL-5): Compounds 4 c (24.0 mg, 0.106 mmol) and 5c (22.2 mg,
0.106 mmol) were used as the starting materials, and KFL-5 was obtained
as a green metallic solid (37.1 mg, 82.8%). Eluent for chromatography:
chloroform. 1H NMR (CDCl3): d=7.83 (dd, J=8.2, 1.3 Hz, 4H), 7.50–
7.42 (m, 6H), 7.12 (s, 1H), 6.96 (s, 2H), 6.53 ppm (s, 2H); 13C NMR
([D6]DMSO, 808C): d=167.6, 152.5, 149.2, 139.3, 130.1, 128.8, 127.9,
125.3, 125.2, 103.9, 95.3 ppm; MALDI-TOF: m/z calcd: 424.1; found:
424.0 [M+].
General procedure for synthesis of 2-subsituted 4H-furoACTHNUTRGNEU[GN 3,2-b]pyrrole-5-
carbaldehydes 5c–5h: Compounds 4c–4h were dissolved in trifluoroace-
tic acid (1 mL) and heated at 508C for 10 min. Triethylorthoformate
(0.5 mL) was added into the reaction mixtures, and stirred for further
10 min. After cooling, the reaction mixture was poured into saturated
aqueous NaHCO3 solution to neutralize. The formed precipitate was fil-
tered, washed with water and dried in vacuo. The precipitate was purified
with flash chromatography (silica gel) to obtain the 2-subsituted 4H-furo-
2,8-Bis(2-methoxyphenyl)-difuroACTHNUTRGENNG[U 2,3-b]ACHTUNGTRNE[NUGN 3,2-g]-5,5-difluoro-5-bora-3a,4a-
diaza-s-indacene (KFL-6): Compounds 4d (44.7 mg, 0.174 mmol) and 5d
(41.0 mg, 0.170 mmol) were used as the starting materials, and KFL-6
was obtained as a green metallic solid (56.7 mg, 67.4%). Eluent for chro-
matography: chloroform. 1H NMR (CDCl3): d=7.98 (d, J=7.8 Hz, 2H),
7.39 (t, J=7.8 Hz, 2H), 7.30 (s, 2H), 7.09–7.00 (m, 5H), 6.46 (s, 2H),
4.01 ppm (s, 6H); 13C NMR ([D6]DMSO, 808C): d=164.2, 163.6, 156.8,
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
(99.7 mg, 0.439 mmol) was used as the starting material, and 5c was ob-
tained as a gray solid (75.9 mg, 81.9%). Eluent for chromatography: n-
hexane/ethyl acetate 75:25!60:40. 1H NMR (CDCl3): d=9.46 (s, 1H),
1104
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1096 – 1106

Fluorescent Dyes
FULL PAPER
139.4, 131.5, 126.5, 120.6, 117.4, 112.0, 103.0, 98.7, 55.6 ppm; MALDI-
2-Methyl-8-phenyl-difuroACTHUNGTRENNU[G 2,3-b]ACHTGNUTREN[NUNG 3,2-g]-5,5-difluoro-5-bora-3a,4a-diaza-s-
TOF: m/z calcd: 484.1; found: 484.0 [M+].
indacene (KFL-12): Compounds 4a (16.0 mg, 0.097 mmol) and 5c
(20.4 mg, 0.094 mmol) were used as starting materials, and KFL-12 was
obtained as a golden metallic solid (15.6 mg, 44.4%). Eluent for chroma-
tography: chloroform. 1H NMR (CDCl3): d=7.80 (d, J=6.9 Hz, 2H),
7.48–7.40 (m, 3H), 7.08 (s, 1H), 6.93 (s, 1H), 6.48 (s, 1H), 6.41 (s, 1H),
6.35 (s, 1H), 2.49 ppm (s, 3H); 13C NMR (CDCl3): d=170.6, 167.6, 153.2,
151.7 149.4, 139.0, 130.0, 129.9, 129.0, 127.3, 125.4, 102.9, 102.5, 98.0, 95.4,
15.9 ppm; MALDI-TOF: m/z calcd: 362.1; found: 362.0 [M+].
2,8-Bis(4-isopropylphenyl)-difuroACTHNUGRTENNUG[2,3-b]ACHTUNGTRNE[NUGN 3,2-g]-5,5-difluoro-5-bora-3a,4a-
diaza-s-indacene (KFL-7): Compounds 4e (26.8 mg, 0.100 mmol) and 5e
(25.1 mg, 0.100 mmol) were used as the starting materials, and KFL-7
was obtained as a green metallic solid (22.5 mg, 44.5%). Eluent for chro-
matography: n-hexane/chloroform 40:60!10:90. 1H NMR (CDCl3): d=
7.75 (d, J=6.8 Hz, 4H), (d, J=7.1 Hz, 4H), 7.05 (s, 1H), 6.91 (s, 2H),
6.46 (s, 2H), 3.01–2.92 (m, 2H), 1.31 (s, 6H), 1.28 ppm (s, 6H); 13C NMR
(CDCl3): d=168.6, 153.5, 151.7, 150.3, 127.4, 127.2, 125.9, 125.7, 102.4,
94.9, 34.2, 29.7, 23.7 ppm; MALDI-TOF: m/z calcd: 508.2; found: 508.1
[M+].
2-(2,4-Dimethoxyphenyl)-7,9-dimethyl-furoACTHNUTRGNEU[GN 2,3-b]-5,5-difluoro-5-bora-
3a,4a-diaza-s-indacene (KFL-13): Compound 4h (29.0 mg, 0.101 mmol)
and 2,4-dimethylpyrrole-5-carbaldehyde (12.5 mg, 0.102 mmol) were used
as starting materials, and KFL-13 was obtained as a golden metallic solid
(16.1 mg, 40.3%). Eluent for chromatography: n-hexane/chloroform
15:85!0:100. 1H NMR (CDCl3): d=7.88 (d, J=9.0 Hz, 1H), 7.12 (s,
1H), 7.02 (s, 1H), 6.59 (dd, J=8.7, 2.4 Hz, 1H), 6.53 (d, J=2.1 Hz, 1H),
6.42 (s, 1H), 6.05 (s, 1H), 3.96 (s, 3H), 3.87 (s, 3H), 2.57 (s, 3H),
2.23 ppm (s, 3H); 13C NMR (CDCl3): d=165.1, 162.5, 158.8, 156.6, 151.1,
140.5, 137.7, 135.2, 128.5, 122.5, 118.8, 112.3, 105.5, 101.8, 98.6, 98.3, 55.6,
55.5, 14.7, 11.3 ppm; MALDI-TOF: m/z calcd: 396.1; found: 396.0 [M+].
2,8-Bis(2-isopropylphenyl)-difuroACTHNUGRTENNUG[2,3-b]ACHTUNGTRNE[NUGN 3,2-g]-5,5-difluoro-5-bora-3a,4a-
diaza-s-indacene (KFL-8): Compounds 4 f (27.5 mg, 0.102 mmol) and 5 f
(26.1 mg, 0.103 mmol) were used as the starting materials, and KFL-8
was obtained as a green metallic solid (24.7 mg, 47.6%). Eluent for chro-
matography: n-hexane/chloroform 40:60!20:80. 1H NMR (CDCl3): d=
7.63 (d, J=7.3 Hz, 2H), 7.48–7.41 (m, 4H), 7.29 (t, J=7.8 Hz, 2H), 7.17
(s, 1H), 6.73 (s, 2H), 6.54 (s, 2H), 3.56–3.49 (m, 2H), 1.30 (s, 6H),
1.26 ppm (s, 6H); 13C NMR (CDCl3): d=169.4, 153.3, 150.5, 148.1, 139.4,
130.5, 129.5, 128.6, 127.5, 126.4, 126.0, 103.2, 99.2, 30.3, 24.1 ppm;
MALDI-TOF: m/z calcd: 508.2; found: 508.1 [M+].
2-(2,4-Dimethoxyphenyl)-8-methyl-difuroACTHNUTRGENN[UG 2,3-b]ACHUTNGTREN[NNGU 3,2-g]-5,5-difluoro-5-
bora-3a,4a-diaza-s-indacene (KFL-14): Compounds 4h (17.1 mg,
0.060 mmol) and 5a (8.8 mg, 0.059 mmol) were used as starting materials,
and KFL-14 was obtained as a green metallic solid (10.7 mg, 42.6%).
Eluent for chromatography: n-hexane/chloroform 10:90. 1H NMR
(CDCl3): d=7.90 (d, J=8.7 Hz, 1H), 7.14 (s, 1H), 7.02 (s, 1H), 6.61 (dd,
J=8.7, 2.4 Hz, 1H), 6.53 (d, J=2.4 Hz, 1H), 6.42 (s, 1H), 6.38 (s, 1H),
6.32 (s, 1H), 3.98 (s, 3H), 3.89 (s, 3H), 2.48 ppm (s, 3H); 13C NMR
([D6]DMSO, 808C): d=171.7, 169.7, 163.6, 158.4, 152.1, 149.7, 147.8,
137.7, 133.4, 127.9, 107.1, 102.7, 99.7, 97.0, 95.0, 56.0, 55.4, 15.0 ppm;
MALDI-TOF: m/z calcd: 422.1; found: 422.0 [M+].
2,8-Bis(2,5-dimethoxyphenyl)-difuroACTHNUTRGENN[UG 2,3-b]ACHUTNGTREN[NUNG 3,2-g]-5,5-difluoro-5-bora-
3a,4a-diaza-s-indacene (KFL-9): Compounds 4g (32.0 mg, 0.111 mmol)
and 5g (30.2 mg, 0.111 mmol) were used as the starting materials, and
KFL-9 was obtained as a green metallic solid (23.6 mg, 39.3%). Eluent
for chromatography: chloroform/ethyl acetate 98:2. 1H NMR (CDCl3):
d=7.51 (t, J=1.5 Hz, 2H), 7.32 (s, 2H), 7.07 (s, 1H), 6.94 (d, J=1.8 Hz,
4H), 6.47 (s, 2H), 3.96 (s, 6H), 3.86 ppm (s, 6H); 13C NMR ([D6]DMSO,
808C): d=168.1, 163.9, 152.5, 138.0, 131.0, 124.1, 117.6, 113.4, 112.7,
111.0, 103.1, 99.7, 98.9, 56.0, 55.5 ppm; MALDI-TOF: m/z calcd: 544.2;
found: 544.1 [M+].
Measurements: All solvents for spectrometry were purchased from
Kanto Chemical. Absorption spectra were recorded on a Hitachi U-2001
double beam spectrophotometer (Hitachi, Tokyo, Japan). Fluorescence
emission spectra and photostabilities were recorded on a F-4500 fluoro-
photometer (Hitachi, Tokyo, Japan) at 258C. Quantum yields were re-
corded on a SREX Fluorolog-3 (Model FL-3–11, Horiba Jobin Yvon,
Kyoto, Japan) at 258C. The instrument was equipped with a R2658P pho-
tomultiplier tube (Hamamatsu Photonics, Shizuoka, Japan) as a fluores-
cence detector. Measurements of quantum yields were performed by
following the method recommended by Horiba Jobin Yvon (see:
http://www.jp.jobinyvon.horiba.com/product_j/spex/quantum_yield/img/
quantum_yields.pdf). A number of diluted solutions of different dye con-
centrations (A<0.10, to prevent reabsorption) were prepared, and the
absorbance (A) and the integrated fluorescence intensity (F) at each con-
centration were recorded. Then a graph of F versus A was plotted to de-
termine the gradient (G). Quantum yields f were calculated by using
Equation (1):
2,8-Bis(2,4-dimethoxyphenyl)-difuroACTHNUTRGENN[UG 2,3-b]ACHUTNGTREN[NUNG 3,2-g]-5,5-difluoro-5-bora-
3a,4a-diaza-s-indacene (KFL-10): Compounds 4h (23.0 mg, 0.080 mmol)
and 5h (21.5 mg, 0.079 mmol) were used as the starting materials, and
KFL-10 was obtained as a green metallic solid (31.2 mg, 71.6%). Eluent
for chromatography: chloroform/ethyl acetate 98:2. 1H NMR (CDCl3):
d=7.90 (d, J=9.0 Hz, 2H), 7.15 (s, 2H), 6.98 (s, 1H), 6.61 (dd, J=9.0,
2.4 Hz, 2H), 6.54 (d, J=2.1 Hz, 2H), 6.40 (s, 2H), 3.98 (s, 6H), 3.89 ppm
(s, 6H); 13C NMR ([D6]DMSO, 808C): d=164.4, 162.5, 158.4, 151.1,
149.4, 139.0, 127.7, 125.7, 110.7, 106.5, 102.1, 98.8, 96.7, 55.7, 55.3 ppm;
MALDI-TOF: m/z calcd: 544.2; found: 544.1 [M+].
General procedure for synthesis of asymmetric KFL dyes (KFL-11–14):
A 2-substituted 4H-furo
solved in trifluoroacetic acid (1 mL) and heated at 508C for 10 min. 2,4-
Dimethylpyrrole-5-carbaldehyde (1 equiv) or 2-substituted 4H-furo[3,2-
ACHTUNGTRENNUNG[3,2-b]pyrrole-5-carboxylic acid (1 equiv) was dis-
AHCTUNGTRENNUNG
b]pyrrole-5-carbaldehyde (1 equiv) and phosphoryl chloride (0.8 mL) was
added into the reaction mixture, and stirred for further 10 min until an
intense color was formed. After cooling, the reaction mixture was poured
into saturated aqueous NaHCO3 solution to neutralize. The formed pre-
cipitate was filtered, washed with water, and dried in vacuo. The resulting
compound was dissolved in 1,1,2-trichloroethane (10 mL), before
BF3·Et2O (0.3 mL) and triethylamine (0.2 mL) were added and the mix-
ture was stirred at 1008C for 15 min. After cooling, the reaction mixture
was diluted with chloroform and washed with saturated aqueous
NaHCO3 solution and brine, dried over Na2SO4 and evaporated. The res-
idue was purified by chromatography (silica gel) to obtain the KFL-11–
14.
ꢀ
ꢁꢀ
ꢁ
GR
GS
nR
nS
ꢀS ¼ ꢀR
ð1Þ
The subscripts R and S denote the reference dye and the sample, respec-
tively, and n is the refractive index of the solvent. The following refer-
ence dyes were used: rhodamine 6G (f=0.95 in ethanol)[34] for KFL-11,
cresyl violet (f=0.54 in methanol)[35] for KFL-1, KFL-2, KFL-12 and
KFL-13, 3,3’-diethyl-thiadicarbocyanine (f=0.35 in ethanol)[36] for KFL-
5, KFL-8 and KFL-14, boronazadipyrromethene compound aza-
BODIPY (f=0.36 in chloroform)[22] for KFL-3, KFL-4, KFL-6, KFL-7,
KFL-9, and KFL-10.
2,7,9-TrimethylfuroACHTUNGTRENNUNG[2,3-b]-5,5-difluoro-5-bora-3a,4a-diaza-s-indacene
(KFL-11): Compound 4a (39.0 mg, 0.236 mmol) and 2,4-dimethylpyrrole-
5-carbaldehyde (29.7 mg, 0.224 mmol) were used as starting materials,
and KFL-11 was obtained as an orange metallic solid (43.2 mg, 66.8%).
Eluent for chromatography: n-hexane/chloroform 20:80!0:100.
1H NMR (CDCl3): d=7.05 (s, 1H), 6.40 (s, 1H), 6.29 (s, 1H), 6.05 (s,
1H), 2.54 (s, 3H), 2.45 (s, 3H), 2.22 ppm (s, 3H); 13C NMR (CDCl3): d=
168.8, 158.0, 152.4, 150.0, 141.8, 136.1, 135.3, 124.1, 119.3, 102.5, 100.6,
97.7, 15.7, 15.6, 11.3 ppm; MALDI-TOF: m/z calcd: 274.1; found: 273.9
[M+].
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Received: September 16, 2008
Published online: December 30, 2008
1106
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Chem. Eur. J. 2009, 15, 1096 – 1106