New repertoire of 'donor-two-acceptor' NIR fluorogenic dyes

Article abstract of DOI:10.1016/j.bmc.2013.02.049

Dye molecules with various fluorescent wavelengths are widely used for diagnostic and optical imaging applications. Accordingly, there is a constant demand for fluorogenic dyes with new properties. We have recently developed a novel strategy for the desig

Full text of DOI:10.1016/j.bmc.2013.02.049

Bioorganic & Medicinal Chemistry 21 (2013) 3602–3608
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New repertoire of ‘donor-two-acceptor’ NIR fluorogenic dyes
⇑
Einat Kisin-Finfer, Doron Shabat
Department of Organic Chemistry, School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 14 March 2013
Dye molecules with various fluorescent wavelengths are widely used for diagnostic and optical imaging
applications. Accordingly, there is a constant demand for fluorogenic dyes with new properties. We have
recently developed a novel strategy for the design of long-wavelength fluorescent dyes with a turn-ON
Keywords:
option. The design is based on a donor-two-acceptor
charge transfer to form a new fluorochrome with an extended
a series of such dyes based on two novel latent donors, naphthol and hydroxycoumarin. One of the dyes
has showed excellent near-infrared fluorescent characteristics and specifically was demonstrated as a
mitochondrial imaging reagent in live cells. This unique strategy for fluorogenic dye design has opened
new doors for further near-infrared fluorescence probe discovery.
p
-electron system that can undergo an internal
-conjugated system. Here, we describe
Fluorescence assay
NIR fluorescence
Turn-ON probes
p
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
remove the protecting group. The p-electron arrangement of phe-
nolate III can be viewed as a general donor-two-acceptor system
that can be modified to prepare probes with a turn-ON option. This
concept was exemplified by preparing a novel NIR turn-ON probe
for detection of hydrogen peroxide. The probe was successfully
used to image induced inflammation in mice.14a
Fluorescent probes are of considerable interest due to their
simple implementation, high sensitivity and high spatial resolu-
tion.1–5 Such probes, based on dye molecules that fluoresce at
various wavelengths, are widely used for diagnostic and optical
imaging applications.6–13 We have recently developed a novel
strategy for the design of long-wavelength fluorogenic dyes with
a turn-ON option.14 Our design is based on a donor-two-acceptor
All of the dyes in our library were based on a phenol donor
moiety with various substituents and acceptor moieties. It is also
possible to use donors with extended conjugation of
p-electron
p
-electron system that can undergo an internal charge transfer to
system. Here we report a new series of donor-two-acceptor
fluorogenic dyes based on naphthol and on hydroxycoumarin
donor nuclei; some have excellent optical qualities.
form a new fluorochrome with an extended -conjugated system.
p
This strategy was demonstrated by synthesis of a library of dyes
that had fluorescence emission in the near-infrared (NIR)
region.15,16,14b This optical range is particularly significant for
in vivo imaging applications, since live tissues have minimal absor-
bance and emission in such wavelengths and the NIR photons have
better penetration abilities.17
Our donor-two-acceptor dye system is composed of a phenol
moiety that functions as a latent donor conjugated with two
acceptors (Fig. 1). Deprotonation of the phenol leads to formation
of a phenolate active donor III that is able to donate a pair of
2. Results and discussion
Three dyes based on a naphthol donor were designed each with
two indolium acceptors (Fig. 2). The dyes were synthesized, using
our previously published procedure14, by condensation of 2 equiv
of the indolium with the corresponded naphthol-dialdehyde.
Dye 1 was synthesized, as outlined in Figure 3, starting from
commercially available 1-naphthol. 1-Naphthol was treated with
hexamethylenetetramine (HMT) in trifluoroacetic acid (TFA) via
the Duff reaction to afford dialdehyde 1a. The latter was condensed
with 2 equiv of indolium 1b to afford dye 1.
p
-electrons to either one of the conjugated acceptors (structures
I and II). This intramolecular charge transfer (ICT) generates a res-
onance species with a -electron pattern similar to that of Cy7
p
fluorochrome. The donor capability of the phenolate species III
can be masked either by a proton or by a specific protecting group.
Such a protected phenol can be used as a molecular probe for
detection or imaging of an analyte that reacts with the probe to
Dye 2 was similarly synthesized starting from compound 2a,
which was treated with hexamethylenetetramine to give dialde-
hyde 2b (Fig. 4). The latter was condensed with 2 equiv of indolium
1b to afford dye 2.
Dye 3 was synthesized as shown in Figure 5. Commercially
available 5,7-dimethyl-tetralone was treated with lithium
diisopropylamine followed by addition of trimethyl silyl chloride
⇑
Corresponding author. Tel.: +972 (0) 3 640 8340; fax: +972 (0) 3 640 5761.
E-mail address: chdoron@post.tau.ac.il (D. Shabat).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.049

E. Kisin-Finfer, D. Shabat / Bioorg. Med. Chem. 21 (2013) 3602–3608
3603
Latent Donor
HO
Acceptor
Acceptor
1
deprotonation
O
Acceptor
Acceptor
III
ICT
ICT
O
O
Acceptor
Donor
Donor
Acceptor
I
II
Figure 1. General mechanism of activation of donor-two-acceptor cyanine-like fluorescence probes.
OH
OH
SO3
N
N
N
SO3
O3S
HO
O3S
SO3
N
N
N
O3S
3
1
2
Figure 2. Chemical structures of donor-two-acceptor dyes composed of naphthol donor and two indolium acceptors.
N
1b
, NaOAc
OH
OH
O3S
CHO
HMT, TFA
71%
Dye 1
Ac2O, 80oC
42%
CHO
1a
Figure 3. Chemical synthesis of dye 1.
N
CHO
HO
1b
HO
HMT, TFA
17%
, NaOAc
O3S
Dye 2
CHO
CHO
Ac2O, 80oC
65%
2a
2b
Figure 4. Chemical synthesis of dye 2.
to afford enol 3a. The latter was oxidized with 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone, followed by deprotection of the
trimethylsilyl group with tetrabutylammonium fluoride to give
naphthol 3b. The naphthol was reacted with pivaloyl chloride to
afford ester 3c, which was then reacted with N-bromosuccinimide
to give dibromide 3d. The dibromide was hydrolyzed in the
presence of silver sulfate to afford diol 3e, which was then oxidized
with manganese oxide to give dialdehyde 3f. The latter was
deprotected with potassium carbonate and then condensed with
2 equiv of indolium 1b to afford dye 3.
The naphthol donor is composed of two fused aromatic rings;
one of the rings has a hydroxyl functional group. Dye 1 has two
indolium acceptors on the phenolic moiety, dye
2 has one
indolium on the phenol ring and the other on the second ring,
and dye 3 has the two indolium acceptors on the non-phenolic
ring. The indolium moieties are located at conjugated positions

3604
E. Kisin-Finfer, D. Shabat / Bioorg. Med. Chem. 21 (2013) 3602–3608
O
OH
O
OTMS
O
tBuC(O)Cl, Et3N, DMAP
1. DDQ, benzene
(iPr)2NH, BuLi, TMS-Cl
2. TBAF, THF
80%
THF, -78oC
96%
DCM
70%
3c
3b
3a
O
O
O
O
MnO2
NBS. (PhCO2)2
HO
Ag2SO4
Br
EtOAc
50%
CCl4
69%
dioxane : water
1
:
1
58%
OH
Br
3e
3d
N
O
O
OH
O3S
1b
OHC
OHC
, NaOAc
1.
K2CO3
Dye 3
MeOH
96%
Ac2O
CHO
CHO
3g
2. K2CO3, MeOH
21%
3f
Figure 5. Chemical synthesis of dye 3.
N
I
N
N
HO
O
N
O
HO
O
O
HO
O
I
O
SO3
SO3
4
6
5
SO3
I
N
N
Figure 6. Chemical structures of donor-two-acceptor dyes composed of hydroxycoumarin donor and indolium or picolinium acceptors.
relative to the phenol donor. Thus, upon deprotonation, the
obtained phenolate can donate an electron-pair through an intra-
molecular charge transfer to either one of the indolium acceptors
and thereby to form donor-two-acceptor dye system, as illus-
trated in Figure 1.
An additional three dyes were prepared based on hydroxy-
coumarin donor with indolium or picolinium acceptors (Fig. 6).
These dyes were synthesized by condensation of the corresponded
dialdehyde with the indolium or picolinium moieties to form
donor-two-acceptor dye systems. Dyes 4 and 5 are composed of
either two picolinium or two indolium acceptors, each on different
ring of the donor. Dye 6 has one picolinium acceptor on the pheno-
lic ring and one indolium on the lactone ring.
The synthesis of dyes 4 and 5 was achieved as outlined in
Figure 7. Coumarin derivative 4a was treated with hexamethylene-
tetramine via the Duff reaction to afford aldehyde 4b. The latter
was reacted with osmium tetroxide in the presence of N-oxide-
N-methyl-morpholine to give diol 4c and then oxidized with
sodium periodate to afford dialdehyde 4d. The dialdehyde was
condensed with 2 equiv of N-methyl-4-picolinium 4e to afford
dye 4. Similarly, dye 5 was prepared by condensation of dialdehyde
4d with 2 equiv of indolium 1b.
Dye 6 was synthesized as outlined in Figure 8. Dialdehyde 4d
was condensed with 1 equiv of picolinium 4e to give compound
6a. The latter was condensed with 1 equiv of indolium 1b to afford
dye 6.
The optical properties and the pKas of the prepared dyes are
summarized in Table 1. All dyes, with the exception of compound
3, exhibited fluorescence emission in the NIR region. This observa-
tion suggested that these dyes have the ability to form a new
donor-two-acceptor fluorochrome after the phenolic species is
converted into a phenolate. Dye 1 absorbance and fluorescence
spectra are presented in Figure 9 (the other dyes spectra can be
found in the Supplementary data). To demonstrate the turn-ON
option of the probe, the spectra were measured under two pH con-
ditions. For evaluation of the protonated form, the measurement
was taken at pH lower than the pKa of the phenol; for analysis of
the deprotonated form, the measurement was taken at pH higher
than the pKa. As expected, a large red-shift was observed in absorp-
tion spectrum when the spectra of the protonated (kmax = 410 nm)
and the deprotonated (kmax = 660 nm) forms were compared.
Importantly, strong fluorescence emission was obtained in the
NIR region for the protonated form, whereas no such fluorescence
was observed for the deprotonated form. Dye 1 exhibited the

E. Kisin-Finfer, D. Shabat / Bioorg. Med. Chem. 21 (2013) 3602–3608
3605
O
N
O
, OsO4
O
O
1. HMT, TFA, reflux
OEt
OEt
DCM: H2O
2. HCl (1M), r.t
54%
1
:
1
HO
O
O
HO
O
O
42%
CHO
4a
4b
Dye 4
N
I
OH
O
CHO
O
OEt
NaIO4 (aq)
, SiO2
OH
HO
O
HO
O
O
DCM
90%
CHO
N
CHO
4c
O3S
4d
Dye 5
Figure 7. Chemical synthesis of dyes 4 and 5.
N
1b
CHO
N
CHO
O
, pyrrolidine
(1eq)
O3S
, piperidine
4e
I
Dye 6
HO
O
I
O
HO
O
EtOH
50%
EtOH
31%
CHO
6a
4d
N
Figure 8. Chemical synthesis of dye 6.
Table 1
Chemical structures and spectroscopic data of donor-two-acceptor based fluorogenic dyes obtained from naphthol or hydroxycoumarin donors
Dye structure
Donor
Acceptors
kex
kem
e
U
(%)
pKa
1
510, 660
740
46,046
7.2
1.95
2
480, 620
750
26,472
1.4
4.25
(continued on next page)

3606
E. Kisin-Finfer, D. Shabat / Bioorg. Med. Chem. 21 (2013) 3602–3608
Table 1 (continued)
Dye structure
Donor
Acceptors
kex
kem
e
U
(%)
pKa
3
400, 570
—
17,391
—
7.65
4
450, 500
650
22,714
5.5
5.43
5
480, 600
700
28,074
16.7
3.8
6
450, 570
670
39,352
12.8
4.42
Figure 9. (Left) Absorption spectra of probe 1, 25
PBS pH 1.0 (red), kex 670 nm, kem 740 nm.
lM in PBS pH 7.4 (blue) and in PBS pH 1.0 (red). (Right) Fluorescence spectra of probe 1, 25 lM in PBS pH 7.4 (blue) and in

E. Kisin-Finfer, D. Shabat / Bioorg. Med. Chem. 21 (2013) 3602–3608
3607
100
80
100
80
60
40
20
60
40
Dye 4 pKa =5.46
Dye1pKa=1.85
20
0
Dye 2pKa=5.35
Dye 3pKa=7.35
Dye 5 pKa =3.80
p
Dye 6 pKa =4.39
0
2
4
6
8
10
0
2
4
6
8
10
pH
pH
Figure 10. Determination of pKa values for the phenols shown in Table 1.
In order to assess which of dyes is most suitable for in vitro and
in vivo imaging, we evaluated their fluorescence stability under
aqueous conditions. The dyes were incubated in PBS (pH 7.4) at
37 °C, and their NIR fluorescent emission was monitored as a func-
tion of time (Fig. 11). Dye 1 had the highest stability under these
conditions of the five dyes evaluated with a T1/2 of over 50 h.
To demonstrate the potential of these dyes to serve as imaging
reagents, we synthesized a probe for mitochondrial imaging. The
synthesis was achieved as outlined in Figure 12. Indolium-acid
7b was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide and hydroxybenzotriazole followed by the addition of com-
pound 7a to give indolium-amide 7c. Two equivalents of this
indolium were condensed with dialdehyde 1a to afford probe 7.
This probe is a derivative of dye 1 linked to a triphenylphospho-
nium moiety, which is known to be internalized by the mitochon-
dria of cells.18 The delocalized positive charge on the lipophilic
triphenylphosphonium cations promotes their mitochondrial
membrane potential dependent accumulation into mitochondria
and their direct passage through phospholipid bilayers. Conse-
quently, these molecules are useful in a range of mitochondrial
studies. DA-3 mammary adenocarcinoma cells were incubated
with the probe and imaged by confocal microscopy. The image in
Figure 13 clearly shows that the mitochondria of these cells are
fluorescent. The design of an analogue with a masked phenol that
will be removed under oxidative stress could be a useful extension
of this example.
Figure 11. Fluorescence stability expressed as the half-life of the indicated dyes in
PBS (pH 7.4) at 37 °C. Each dye was excited at the wavelength given in Table 1.
longest excitation/emission wavelength-pair of the dyes prepared
based on the donor-two-acceptor design.
The relatively large shift in absorption spectrum between
protonated and the deprotonated forms of these dyes allows
convenient measurement of the pKa values of the dyes. The pKas
of the six dyes were measured by monitoring the kmax of their
UV–vis absorbance as
a function of the environmental pH
(Fig. 10). The relative low pKa values obtained for the phenolic dyes
indicate that under physiological conditions, these dyes are in the
phenolate form. Under typical intracellular conditions, the
intramolecular-charge-transfer mode of action will occur, and the
dyes will emit NIR fluorescence.
The described dye molecules are composed of a phenol latent
donor conjugated with two acceptors. The latent donor is turned
ON upon formation of a phenolate ion. This unique design enables
an intramolecular charge transfer from the activated donor to one
N
OH
Br
CHO
OH
CHO
7b
, NaOAc
N
N
1a
PPh3
, EDC, HOBt
COOH
Br
N
H2N
Br
DMF, 54%
Ac2O
17%
Br
Br
7a
7
7c
O
O
O
NH HN
NH
Ph3P
Br
PPh3
Br
Ph3P
Br
Figure 12. Chemical synthesis of mitochondrial imaging probe 7.

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E. Kisin-Finfer, D. Shabat / Bioorg. Med. Chem. 21 (2013) 3602–3608
Figure 13. Mitochondrial imaging using probe 7 in cultured mouse DA-3 mammary adenocarcinoma cells (ex/em, 635 nm/710 nm).
of the two acceptors to form
chromophore between the former two acceptors. The newly
formed push–pull structural element has a larger -system than
a
new push–pull conjugated
(GIF) for financial support. This work was supported in part by
Grants from the Israeli National Nanotechnology Initiative and
Helmsley Charitable Trust for a focal technology area on Nanomed-
icines for Personalized Theranostics. D.S. thanks Professor Ilan
Tsarfaty and Miriam Shaharabany for supplying the confocal image
of Figure 13.
p
that of the original donor–acceptor and thus can emit fluorescence
at longer wavelengths. The introduction of naphthol or of hydroxy-
coumarin as the donor moiety has extended our repertoire of
fluorogenic probes. Five of the six new dyes demonstrated good
optical characteristics with fluorescence emission in the NIR
region. Interestingly dye 3 was the only one that failed to fluoresce,
probably, due to the absence of an acceptor moiety on the phenolic
ring. Indeed phenol 3 had the highest pKa value among the
prepared dyes, and although a visible red-shift was observed in
the deprotonated form, no fluorescence was obtained.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.02.
049.
References and notes
3. Conclusions
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probes. The design is based on a donor-two-acceptor
system that can undergo an intramolecular charge transfer to form
a new fluorochrome with a longer -conjugated system. Two dif-
p-electron
p
ferent latent donors, naphthol and hydroxycoumarin were conju-
gated with indolium or picolinium acceptors into the modular
probe platform to generate versatile dye compounds. One of the
dyes, which had significant stability at physiological pH in aqueous
conditions, was conjugated with a lipophilic cation that is readily
taken up by mitochondria. Confocal images of DA-3 mammary
adenocarcinoma cells incubated with this probe indicated that
the probe penetrates cells and can be used as mitochondrial
imaging reagent. This unique strategy for dye design has opened
a new doorway for further NIR fluorescence probe discovery.
16. Karton-Lifshin, N.; Presiado, I.; Erez, Y.; Gepshtein, R.; Shabat, D.; Huppert, D. J.
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Acknowledgments
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D.S. thanks the Israel Science Foundation (ISF), the Binational
Science Foundation (BSF) and the German Israeli Foundation