Cyanine dye conjugates as probes for live cell imaging

Article abstract of DOI:10.1016/j.bmcl.2007.06.097

A series of fluorescent compounds suitable for live cell imaging is described. Functionalized forms of four different asymmetric cyanine dyes are reported that are amenable to peptide conjugation. The photophysical properties of the modified dyes and conj

Full text of DOI:10.1016/j.bmcl.2007.06.097

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5182–5185
Cyanine dye conjugates as probes for live cell imaging
Jay R. Carreon,b Kelly M. Stewart,a Kerry P. Mahon Jr.,b
Stephanie Shinb and Shana O. Kelleya,*
aUniversity of Toronto, Department of Biochemistry, Toronto, Ont., Canada M5S 1A8
bBoston College, Eugene F. Merkert Chemistry Center, Chestnut Hill, MA 02467, USA
Received 11 May 2007; revised 21 June 2007; accepted 28 June 2007
Available online 10 July 2007
Abstract—A series of fluorescent compounds suitable for live cell imaging is described. Functionalized forms of four different asym-
metric cyanine dyes are reported that are amenable to peptide conjugation. The photophysical properties of the modified dyes and
conjugates and the use of the compounds as cellular imaging agents are described. The results obtained indicate that these spectrally
versatile compounds, which have absorption and emission profiles spanning the visible spectrum, are useful probes for cellular
imaging.
Ó 2007 Elsevier Ltd. All rights reserved.
Fueled by advancements in imaging technology, exami-
nation of live cells by fluorescence techniques is becom-
ing widely used to study cellular processes.1
Fluorophores that are permeable to intact cell mem-
branes provide an opportunity to perform dynamic anal-
yses in living cells and also avoid limitations associated
with fixation. In contrast to fixed cells, which only pro-
vide a snapshot of a biological sample, visualization of
cellular processes in living cells with techniques such as
time-lapse microscopy and flow cytometry allows for
real-time analysis. In addition, the process of fixation
has been shown to alter the localization of some mole-
cules, which can lead to experimental artifacts.2,3 While
a limited number of dyes for live cell analysis are avail-
able commercially, new cell-permeable fluorophores with
versatile spectroscopic properties are needed to provide
additional options for live cell analysis.
of compounds exhibits high molar extinction coefficients
permitting the use of low concentrations. These prop-
erty, along with high fluorescence quantum yields, make
these molecules ideal cellular fluorophores. Although
cyanine dyes offer many valuable properties as a cellular
imaging tools, those that are commercially available for
cell-based studies (e.g., TO-PRO, TO3-PRO) can only
be used as an imaging tool with fixed cells as they are
unable to penetrate an intact cell membrane.9 In addi-
tion, these commercially available derivatives are not
amenable to biomolecule conjugation. Herein, we report
the synthesis of four cyanine dye derivatives that can be
easily functionalized with peptides or other biomole-
cules. In addition, we report on the properties of a set
of designed cyanine dye–peptide conjugates that serve
as live cell mitochondrial markers.
Four cyanine dye derivatives were synthesized. The syn-
thetic approach is based on one described previously for
small-scale solid-phase synthesis of modified cyanine
dyes.10 The approach developed here is amenable to
the production of much larger quantities of the com-
pounds (the syntheses described here produce 50–90%
yields on gram quantities of starting material), and the
intermediates can be completely characterized. The dyes
made contain either a pyridinium or quinolinium ring
linked to a benzothiazolium ring via a monomethine
(4, 5) or trimethine linker (6, 7).
Cyanine dyes, a class of synthetic fluorophores with two
heteroaromatic rings joined by a monomethine or
polymethine chain, have been used for a variety of bio-
logical applications requiring spectroscopic labels.4–9
These dye molecules have tunable wavelengths across
the visible spectrum, which allows for spectral compati-
bility in multicolor applications. In addition, this family
Keywords: Peptide conjugate; Fluorescence imaging; Cellular imaging;
Dyes.
Monomethine cyanine dye derivatives were prepared as
shown in Scheme 1. The benzothiazole derivative 1a was
*
Corresponding author. Fax: +1 416 978 2979; e-mail:
shana.kelley@utoronto.ca
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.097

J. R. Carreon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5182–5185
5183
O
R
1. HBTU, i-Pr2NEt,
S
O
Me
N
DMF, rt, 3 h
n
5
4a-7a H2N
FrFK
N
N
Me
FrFK-CONH2
2. TFA:TIS (95:5),
rt, 1 h
2a or 2b
5
Me
N
O
S
R
4c n = 0, BO
5c n = 0, TO
6c n = 1, BO3
7c n = 1, TO3
N
5
Me Et3N, DCM
4a R = OH
4b R = OMe
S
N
S
rt, 24 h
Scheme 3. Solid-phase synthesis of dye–peptide conjugates 4c–7c.
Me
Me
N
O
R
1a
S
N
5
using standard solid-phase Fmoc chemistry, and the
resulting peptide conjugate cleaved from the resin
(Scheme 3). Purification via HPLC afforded dye–peptide
conjugates 4c–7c. The dye conjugates were then charac-
terized using ESI mass spectrometry, UV–vis spectros-
O
R
5a R = OH
5b R = OMe
Me
N
5
3a or 3b
copy,
and
fluorescence
spectroscopy.
The
Scheme 1. Synthesis of monomethine cyanine dye derivatives 4 and 5.
characterization methods and spectral data are de-
scribed in the Supplementary information.
prepared from 3-methylbenzothiazole-2-thione. Con-
densation of 1a with pyridinium (2a–2b) or quinolinium
(3a–3b) derivatives in the presence of triethylamine in
DCM afforded the carboxylic acid monomethine dyes
4a and 5a or methyl ester dyes 4b and 5b.
These cyanine dyes are frequently utilized as nucleic acid
stains, as their fluorescence quantum yield increases
upon nucleic acid binding or other environmental
changes that rigidify the chromophore.12,13 In order to
determine how the modified dyes and peptide conjugates
performed as nucleic acid stains, and to characterize
their spectroscopic properties for cellular imaging, we
investigated the effect of complexation to double-
stranded DNA on the spectroscopic properties of the
dye-based conjugates. The modified dyes were examined
as methyl esters so that the acidic terminus of the tether
would not interfere with DNA binding. Absorption
spectra of the dye–peptide conjugates (4c–7c) are shown
in Figure 1. The attachment of the peptide sequence to
the dyes causes a slight red-shift of the absorption max-
ima relative to the dye methyl esters (4b–7b). All of the
dye–peptide conjugates displayed a DNA-dependent
enhancement in fluorescence as the methyl esters that
is presumably due to rigidification of the heterocyclic
ring system upon intercalation (Table 1). Interestingly,
BO3 (6) exhibits a unique property compared to the
other three dyes, having a significant fluorescence quan-
tum yield when free in solution (see Supplementary
Trimethine cyanine dye derivatives were synthesized as
shown in Scheme 2. The benzothiazole derivative 1b
was prepared in two steps from 2-methylbenzothiazole.
Condensation of 1b with pyridinium (2b) or quinolinium
(3a–3b) derivatives in the presence of triethylamine in
DCM then yielded the trimethine dyes functionalized
as its carboxylic acid (7a) or methyl ester (6b and 7b).
We were unable to prepare carboxy-functionalized dye
6a using this method; instead, saponification of the
methyl ester 6b with LiOH in MeOH/H2O was used to
produce 6a.
Next, cyanine dye–peptide conjugates were prepared
using the carboxy-functionalized dyes. These conjugates
feature a tetrapeptide sequence with alternating aro-
matic and basic amino acids, Phe-d-Arg-Phe-Lys
(FrFK), a sequence known to facilitate mitochondrial
localization in cultured human cells.11 The peptide seg-
ments of the dye conjugates 4c–7c were prepared on
Rink amide solid support, the N-terminus deprotected
and coupled to the carboxy-functionalized dyes 4a–7a
1
O
R
0.8
Me
N
5
Me
N
2b
0.6
S
O
TO3
BO
(7c)
(4c)
R
N
5
Ac
LiOH
MeOH:H2O
rt, 2 h
6a R = OH
0.4
N
S
N
Et3N, DCM
rt, 24 h
Ph
BO3
(6c)
TO
(5c)
6b R = OMe
Me
1b
0.2
0
Me
N
S
O
R
O
N
5
R
300
400
500
600
700
800
Me
N
5
λ (nm)
7a R = OH
7b R = OMe
Figure 1. Normalized absorption spectra of the cyanine dye peptide
conjugates (4c–7c) in H2O; (4c) BO (solid line); (5c) TO (short dash);
(6c) BO3 (long dash/short dash); (7c) TO3 (long dash/dots).
3a or 3b
Scheme 2. Synthesis of trimethine cyanine dye derivatives 6 and 7.

5184
J. R. Carreon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5182–5185
Table 1. Fluorescence quantum yields (U) for dye–peptide conjugatesa
Compound
kmaxAbs (nm)
kmaxEm (nm)
Urel (DNA)
Enh.d
BO-CO2Me (4b)
BO-FrFK (4c)
455
458
511
512
564
567
634
638
480
480
525
526
593
594
0.029 (2)b
0.069 (1)b
0.141 (4)b
0.206 (2)b
0.093 (1)c
0.116 (2)c
0.024 (3)c
0.008 (2)c
120
140
470
430
8
TO-CO2Me (5b)
TO-FrFK (5c)
BO3-CO2Me (6b)
BO3-FrFK (6c)
TO3-CO2Me (7b)
TO3-FrFK (7c)
4
574, 649
573, 655
20
6
a See Supplementary information for methods used to obtain values tabulated.
b Measured in samples containing 1.5 lM dye, 45 lM bp CT DNA and reported relative to a fluorescein standard.
c Measured in samples containing 1.5 lM dye, 45 lM bp CT DNA and reported relative to a rose bengal standard.
d Fluorescence enhancement observed in presence of DNA relative to buffered solution calculated using values tabulated in Table S1.
information). In general, the emission profiles were not
strongly affected by the presence of the appended pep-
tide, and the emission enhancements observed in the
presence of DNA were similar for the dyes and the
dye–peptide conjugates.
In order to test the utility of this series of cyanine dyes
for cellular imaging, the two sets of compounds (i.e.,
the peptide conjugates and the dye precursors displaying
the esterified tether) were incubated with HeLa cells and
cellular localization was monitored by confocal laser
scanning microscopy (CLSM). As shown in Figures 2
and 3, the four cyanine dyes were each able to cross
the plasma membrane when the terminal carboxylic acid
moiety was either capped as a methyl ester or conju-
gated to the N-terminus of a tetrapeptide. All four cya-
nine methyl esters (4b, 5b, 6b, 7b) rapidly entered living
cells and 4b, 6b, and 7b primarily accumulated in the
mitochondria (Fig. 2). Many lipophilic cations are
known to accumulate in the mitochondria, and the cya-
nine dyes synthesized here appear to adhere to this
trend. Compound 4b also exhibited a limited extent of
nucleolar staining. While cells treated with 4b, 6b, and
7b remained viable over the course of imaging experi-
ments, incubation with 5b resulted in cytotoxicity after
Figure 3. Fluorescence images of (A) BO-FrFK (4c); (B) TO-FrFK
(5c); (C) BO3-FrFK (6c); (D) TO3-FrFK (7c); HeLa cells were
incubated with 5 lM conjugate at 37 °C/5% CO2/humidity for 90 min
and washed with PBS. The cells were then visualized by CLSM using
an excitation wavelength of 488 nm for A, B; 543 nm for C; 633 nm for
D.
a short incubation time (15 min) (see Fig. S10). The on-
set of cell death and resultant perturbation of the mito-
chondrial potential may therefore be responsible for the
nuclear staining seen for this compound.
The peptide conjugates of the modified cyanine dyes are
also useful mitochondrial dyes and exhibit more specific
localization than the parent dyes. As shown in Figure 3,
mitochondrial localization was exclusively observed
when the peptide sequence Phe-d-Arg-Phe-Lys-NH2
was appended. Uptake was reduced, which necessitated
longer incubations and higher concentrations of dye, but
no toxicity was observed (Fig S11). These conjugates are
therefore more effective mitochondrial stains than the
parent dyes and are more appropriate for live cell imag-
ing. Moreover, the spectroscopic characteristics of the
parent dye are retained upon conjugation, thereby
allowing for the visualization of unfixed mammalian
cells using these peptide derivatives using common exci-
tation and emission ranges.
Figure 2. Fluorescence images of (A) BO (4b); (B) TO (5b); (C) BO3
(6b); (D) TO3 (7b); HeLa cells were incubated with 1 lM conjugate at
37 °C/5% CO2/humidity for 15 min and washed with PBS (1x). The
cells were then visualized by CLSM using an excitation wavelength of
488 nm for A, B; 543 nm for C; 633 nm for D.
We have synthesized a series of novel dye–peptide con-
jugates that bind nucleic acids and label live human
cells. These dyes provide a practical method to visualize

J. R. Carreon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5182–5185
5185
the mitochondria of living cells using a variety of obser-
vation wavelengths with excitation in the visible range.
The straightforward syntheses of these dye molecules
feature a carboxylic acid tether that can easily be conju-
gated to a peptide sequence, as demonstrated here, or to
other biomolecules of interest. Importantly, the cytotox-
icity of this type of compound can be reduced by utiliz-
ing standard solid-phase peptide synthesis to attach a
tetrapeptide sequence. Our results indicate that these
untethered dyes and their peptide conjugates can be
used to probe living cells, broadening the application
for this class of fluorophores.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.06.097.
References and notes
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We wish to acknowledge the following sources for sup-
port of this work: the Sloan Foundation for a Research
Fellowship to S.O.K., the Dreyfus Foundation for a Ca-
mille Dreyfus Teacher-Scholar Award to S.O.K., the
NSF for a CAREER award to S.O.K and the Beckman
Foundation for a Beckman Scholars Fellowship to S.S.
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Supplementary data
Experimental procedures and full characterization of
compounds 1a–7c. as well as DIC images of HeLa cells
treated with 4b,c–7b,c. This material is available free of
charge via the Internet at http://www.elsevier.com.