Twisted cyanines: A non-planar fluorogenic dye with superior photostability and its use in a protein-based fluoromodule

Article abstract of DOI:10.1021/ja308629w

The cyanine dye thiazole orange (TO) is a well-known fluorogenic stain for DNA and RNA, but this property precludes its use as an intracellular fluorescent probe for non-nucleic acid biomolecules. Further, as is the case with many cyanines, the dye suffer

Full text of DOI:10.1021/ja308629w

Article
pubs.acs.org/JACS
Twisted Cyanines: A Non-Planar Fluorogenic Dye with Superior
Photostability and its Use in a Protein-Based Fluoromodule
†
†
‡,§
†,§,
Nathaniel I. Shank, Ha H. Pham, Alan S. Waggoner, and Bruce A. Armitage *
†
‡
§
Departments of Chemistry and Biological Sciences and Molecular Biosensor and Imaging Center, Carnegie Mellon University,
400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States
4
*
S Supporting Information
ABSTRACT: The cyanine dye thiazole orange (TO) is a well-
known fluorogenic stain for DNA and RNA, but this property
precludes its use as an intracellular fluorescent probe for non-
nucleic acid biomolecules. Further, as is the case with many
cyanines, the dye suffers from low photostability. Here, we
report the synthesis of a bridge-substituted version of TO named
α-CN-TO, where the central methine hydrogen of TO is
replaced by an electron withdrawing cyano group, which was
expected to decrease the susceptibility of the dye toward singlet
oxygen-mediated degradation. An X-ray crystal structure shows
that α-CN-TO is twisted drastically out of plane, in contrast to
TO, which crystallizes in the planar conformation. α-CN-TO
retains the fluorogenic behavior of the parent dye TO in viscous
glycerol/water solvent, but direct irradiation and indirect
bleaching studies showed that α-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted
conformation of α-CN-TO mitigates nonspecific binding and fluorescence activation by DNA and a previously selected TO-
binding protein and exhibits low background fluorescence in HeLa cell culture. α-CN-TO was then used to select a new protein
that binds and activates fluorescence from the dye. The new α-CN-TO/protein fluoromodule exhibits superior photostability to
an analogous TO/protein fluoromodule. These properties indicate that α-CN-TO will be a useful fluorogenic dye in combination
with specific RNA and protein binding partners for both in vitro and cell-based applications. More broadly, structural features
that promote nonplanar conformations can provide an effective method for reducing nonspecific binding of cationic dyes to
nucleic acids and other biomolecules.
INTRODUCTION
dyes toward dsDNA in solution can be attributed in part to
■
electrostatic attraction but more significantly to the ability of
The number of fluorescent labels used for biological imaging
has grown significantly in the past few decades, driven by the
desire for multicolor labeling, increased sensitivity and
1
2,13
the dye to intercalate between adjacent base pairs.
This
interaction produces stabilizing π-stacking interactions and
mitigates unfavorable hydrophobic interactions with the
aqueous environment. To this end, cyanine dyes can be used
as dead cell stains and in solution or in gels to detect DNA. In
fact, cyanines have been reported to be able to detect DNA at
1
−5
functional analyses.
A number of factors are considered in
developing new fluorescent labels. Ultimately, the aptitude of
any label can be measured by its brightness (ε x ϕ ), lack of
F
background fluorescence/nonspecific localization, ability to
withstand prolonged exposure to light and redox stresses,
overall size, and ease of covalent attachment or noncovalent
binding to the target of interest.
Cyanine dyes are fluorescent molecules that embody a
number of desirable qualities such as high extinction coefficient,
tunable absorption/emission spectra, ease of synthesis and a
10
picogram quantities. Other researchers have developed RNA
aptamers capable of binding nonintercalating fluorogenic
14−17
dyes.
This could allow for the labeling of specific RNA
molecules without concomitantly labeling any double stranded
DNA present, as demonstrated most elegantly by the recently
reported RNA aptamers that bind fluorophores based on green
18
6
fluorescent protein. Further, when not specifically bound by a
single biomolecule, properly functionalized cyanine dyes can be
noncovalently incorporated into the membranes of cells or
organelles and are able to report on changes within the local
moderate-to-high quantum yield. Over the years, these dyes
have been employed as covalent and noncovalent labels.
Cyanine dyes have been used as covalently attached fluorescent
7
,8
labels since 1989 and still see widespread use in labeling
19
9
environment.
applications. Noncovalent labeling methods have seen an even
longer history in fluorescence spectroscopy and imaging. A
well-known example of noncovalent labeling is the staining of
Received: August 30, 2012
1
0,11
double-stranded DNA.
The high affinity of certain cyanine
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ja308629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Chart 1. Fluorogenic Cyanine Dye Structures
Scheme 1
Recently, dye-binding proteins have been developed to
exploit the fluorogenic properties of cyanines and similar
compounds in an effort to make genetically encodable
fluorogenic protein tags. For example, trans-stilbene undergoes
nonradiative excited state deactivation by isomerization,
resulting in a low fluorescence quantum yield in nonviscous
and cyanine dyes. Of the reported modifications, the attach-
ment of an electron withdrawing cyano group in the methine
bridge at the α position has proven to be particularly useful,
with several-fold increases in photostability when introduced
2
7,31
into cyanines having extended polymethine bridges.
This
motivated the synthesis and characterization of a new, cyano-
substituted version of thiazole orange, described herein.
2
0,21
solution.
However, binding of trans-stilbene to an antibody
selected against a stilbene-hapten results in up to 40-fold
2
2−24
fluorescence enhancement.
Similarly, fluorogenic triphe-
RESULTS
■
nylmethane and cyanine dyes have been used to select single
Dye Synthesis. Chemical synthesis of α-CN-TO was
accomplished in a total of five steps in an overall yield of
0% (Scheme 1; the alternative route, in which the central
methine carbon and cyano group are provided by the quinoline
heterocycle was unsuccessful). This approach requires the
redesign of the synthetic route usually followed to create TO
dyes, which typically involves placing the leaving group on the
benzothiazole half-dye. Quaternization of the commercially
available 2-methylthio-benzothiazole readily gave I, which
reacts with cyanoacetic acid in the presence of base to undergo
chain variable fragment (scFv) proteins to create dye−protein
2
5
pairs called fluoromodules. In the original report by Szent-
2
Gyorgyi et al., a naiv
̈
e library of yeast displaying roughly 109
different recombinant human scFvs was challenged with either
malachite green (MG) or thiazole orange (TO) to select clones
capable of binding and fluorescently activating the comple-
2
5
mentary dye.
The ability to rationally design fluorogenic cyanine dyes to
exhibit fluorescence in virtually any desired color and the
powerful selection method based on fluorescence-activated cell
sorting used to identify protein partners has resulted in a
diverse catalogue of fluoromodules in which the dye-protein
32
a malonic ester type reaction at the α carbon. Spontaneous
decarboxylation leads to the precipitation of the final half-dye
II) from aqueous solution. The commercially available 4-
(
dissociation constant (K ) values are generally in the low-mid
nanomolar range and the fluorescence quantum yields can be
D
hydroxyquinoline was first converted to 4-chloroquinoline (III)
in refluxing POCl3 then quaternized with iodomethane.
Electrospray ionization data showed the presence of the
intended N-methyl-4-chloroquinolinium salt (IV) as well as
the 4-iodo analogue, but both species should readily undergo
nucleophilic aromatic substitution, so the chloro-product was
not purified further. The two half-dyes were then condensed at
180 °C to give α-CN-TO as a red solid.
25−28
exceptionally high (up to ϕ = 1.0).
To further increase the
f
utility of these fluoromodules, recent efforts have focused on
improving their photostability. Direct modifications to cyanine
2
9,30
dyes with electron-withdrawing fluorine
(e.g., TO-4F in
(e.g., α-CN-DIR in Chart 1) have
been used in the past to increase the stability of merocyanine
2
7,31
Chart 1) or cyano groups
B
dx.doi.org/10.1021/ja308629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Figure 1. Absorbance and fluorescence spectra of 1 μM TO (A, B) and 2 μM α-CN-TO (C, D) in methanol, sodium phosphate buffer (pH 7.4) or
9
0% glycerol in water. Samples were excited at 480 nm.
Spectroscopic Characterization. We first explored the
spectra of TO and α-CN-TO in methanol, aqueous buffer, and
90% glycerol-in-water, a viscous medium. Whereas α-CN-TO is
not as bright as TO (ca. 4-fold less based on the absorbance-
normalized, integrated spectra), this result shows that the new
dye is fluorogenic. Moreover, whereas TO exhibits residual
fluorescence in buffer, α-CN-TO is absolutely dark.
effects of the cyano substitution on the dye’s absorption
spectrum in homogeneous solutions (methanol, aqueous buffer,
and 90% glycerol in water). It was found that the absorption
band of α-CN-TO had a slight hypsochromic shift of about 16
nm compared to TO under all of the different solvent
conditions (part A vs C of Figure 1). Previously reported dyes
having an α-CN substitution showed much larger shifts of 40−
Photostability. To assess whether or not the cyano
substitution bestows the same photostability for a mono-
methine dye as previously seen with trimethine dyes, we used a
series of bleaching studies to compare α-CN-TO and TO in
viscous (90% glycerol) and nonviscous solutions (buffer). In
the first set of experiments, we irradiated each dye with white
light filtered through a 380 nm long-pass filter. After 60 min of
irradiation the absorbance of TO decreases by ca. 69%, whereas
the spectrum of α-CN-TO was essentially unchanged (parts A
and C of Figure 2). Even after accounting for the higher molar
absorptivity of TO, this is a striking difference. The same
experiment was next repeated in 90% glycerol. Similar to the
previous experiment in nonviscous solution, TO proved to be
significantly less stable than α-CN-TO bleaching by ca. 23%
after 60 min (part B of Figure 2), whereas α-CN-TO exhibited
no loss of fluorescence (part D of Figure 2). The higher
photostability of TO in glycerol compared with buffer is likely
due at least in part to the higher fluorescence quantum yield in
the more viscous solvent. Another factor that could be
contributing to this result is the concentration of dissolved
oxygen, a major player in the photobleaching of cyanine dyes in
the form of singlet oxygen generated by the dye triplet state.
The concentration of oxygen is estimated to drop by 20% for a
6
0 nm but those dyes had longer conjugated systems than
2
7,31
TO.
Similar to other cyano substituted dyes, the extinction
coefficient of α-CN-TO decreased relative to the parent dye, in
−1
−1
this case by approximately half (εmax = 32,500 M cm in
methanol). Although the extinction coefficient is decreased by
α-CN modification, the modest blue-shift improves the new
dye’s overlap with the commonly used 488 nm laser line. In all
three solvents, the absorption spectrum of α-CN-TO does not
exhibit the vibrational structure normally found as a short-
wavelength shoulder for monomethine cyanine dyes such as
TO.
The fluorescence properties of α-CN-TO were investigated
in the same solutions. Similar to other unsymmetrical cyanines,
α-CN-TO exhibited very low background fluorescence in
3
0,33
methanol and aqueous solutions.
This property is
attributed to the ability of cyanine dyes to dissipate absorbed
energy by rotating about the central methine carbon(s) to
adopt a twisted conformation that relaxes nonradiatively to the
3
0,34
ground state.
However, in viscous solvents or if conforma-
tionally restrained, the dyes’ ability to exploit this relaxation
route to return to the ground state is impeded leading to an
increase in other deactivation pathways, most notably
fluorescence. Parts B and D of Figure 1 show the fluorescence
35
∼14% glycerol solution and as much as 75% for pure
36
glycerol. Other factors, such as singlet oxygen lifetime and
C
dx.doi.org/10.1021/ja308629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Figure 2. Photobleaching studies of TO and α-CN-TO. Absorbance spectra of 1 μM TO (A, B) and 2 μM α-CN-TO (C, D) in sodium phosphate
buffer (pH 7.4) (A, C) or 90% glycerol in water (B, D) recorded before and after 60 min irradiation with visible light (λ > 380 nm).
Figure 3. Time course for the bleaching of TO (A) and α-CN-TO (B) in the presence of singlet oxygen generated from molybdate and hydrogen
peroxide. The rising absorption seen at short wavelengths is due to H O . Reactions were run in water with [dye] = 1 μM and pH 9; [Na MoO ] =
2
2
2
4
5
0 mM, [H O ] = 9.8 mM.
2 2
diffusion could also contribute to the difference in bleaching
between these two solutions.
bleached by 82% after 60 min while α-CN-TO showed no
apparent change in its UV−vis spectrum (Figure 3). Upon the
basis of all of the bleaching studies, it is evident that α-CN-TO
exhibits impressive photostability and is completely resistant to
oxidative attack by singlet oxygen.
DNA Binding. As mentioned previously, a number of
biomolecules, most notably DNA, are capable of binding and
activating the fluorescence of TO. It was therefore surprising to
discover that α-CN-TO gave no fluorescence in the presence of
DNA while TO shows a marked increase in its signal (Figure
4). Typically, to achieve such a large apparent reduction in
Singlet Oxygen Reactivity. Assuming the photobleaching
of TO is due at least in part to singlet oxygen, the greater
photostability of α-CN-TO could indicate lower reactivity
toward singlet oxygen for the substituted dye, as we observed
27
previously for α-CN-DIR. To explore the latter pathway, we
performed thermal bleaching studies on both dyes in the
presence of molybdate ions, which catalytically break down
hydrogen peroxide to form water and singlet oxygen via a
37−39
nonphotochemical pathway.
Under these conditions TO
D
dx.doi.org/10.1021/ja308629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Cell Staining. To check for nonspecific staining of
endogenous DNA or cellular components, we further screened
both dyes against mammalian cells. Keeping the excitation
intensity and detector settings fixed we imaged HeLa cells at 1
μM dye concentration without washing. Figure 6 shows that
TO not only stains nuclear DNA but is also fluorescent in the
mitochondria, whereas α-CN-TO shows negligible fluorescence
for any cellular component. Further, the fluorescence signal for
TO continues to accumulate in these areas with time (Figure
S1 of the Supporting Information), whereas the images with α-
CN-TO remain dark. Although it is possible that the lack of
cellular fluorescence α-CN-TO is due to poor uptake of this
dye by HeLa cells, this seems unlikely given the fact that both
TO and α-CN-TO are monocations and have similar molecular
weights. It is also noteworthy that α-CN-TO does not exhibit
any obvious cytotoxic effects at low micromolar concentration.
X-ray Crystallography. The large discrepancy in apparent
binding affinities for TO and α-CN-TO toward DNA led us to
hypothesize that the cyano group was causing the heterocycles
to be twisted out of plane at the central methine carbon
because a nonplanar dye conformation would hinder
intercalation between the DNA base pairs. Typically,
monomethine cyanine dye conformations can be determined
in solution using 2D NMR techniques, particularly observation
of a nuclear Overhauser effect between the central methine
proton and the quinoline ring system. However, the lack of a
proton on the methine carbon in α-CN-TO precludes this
approach. Fortunately, we were able to obtain crystal structures
of both α-CN-TO and TO at a resolution of 0.83 Å (Figure 7).
As expected, the heterocycles of TO are nearly planar and the
dye adopts a conformation that projects the larger part of the
quinoline system away from the sulfur heteroatom of the
benzothiazole system (as drawn in Chart 1). A similar structure
was reported recently for a TO derivative N-functionalized with
Figure 4. Fluorescence spectra of fluorogenic dyes (1 μM) in presence
of calf thymus DNA (50 μM base pairs). Samples were excited at 480
nm.
affinity toward dsDNA, sterically bulky groups or negatively
charged residues that provide electrostatic repulsion are
1
7,28
installed on cyanine dyes.
Here, however, a simple bridge
substitution completely eliminates the typical unsymmetrical
cyanine fluorescence response. We reasoned that this lack of
signal could be due to failure of α-CN-TO to intercalate into
DNA as TO does or, a less likely but plausible theory, the dye is
bound but in a conformation that is unfavorable for
fluorescence.
We tested the possibility that α-CN-TO was binding DNA in
a nonfluorescent manner by performing a competition assay.
Either TO or α-CN-TO was titrated into a solution of DNA in
the presence of TO-3 (Chart 1), the trimethine version of TO.
TO-3 is a monocationic version of the dye TO-PRO-3 that is
41
a propanoic acid substituent on the quinoline ring. In stark
contrast, α-CN-TO is severely twisted with a dihedral angle
between the two heterocycles of about 135° (i.e., the benzene
component of the quinoline system is now closer to the
benzothiazole sulfur). The inability of the nonplanar α-CN-TO
to bind DNA is similar to the behavior of cyanine dyes based
on dimethylindole heterocycles, where the steric bulk of the
geminal methyl groups prevents intercalation. In addition, the
nonplanar structure of α-CN-TO can be envisioned to render
the dye more conformationally flexible than the planar TO,
40
known to bind to DNA. As shown in Figure 5, an incremental
decrease in the emission of TO-3 is seen as TO is titrated into
the solution. However, when α-CN-TO is titrated into the
solution of DNA/TO-3, the emission spectrum of TO3 is
minimally affected. These results indicate that TO can displace
TO-3 from DNA but α-CN-TO cannot. Thus, it is likely that
the lack of fluorescence for α-CN-TO in the presence of DNA
is due to lack of binding of the dye to the DNA.
Figure 5. Competition assays between 6 μM base pairs ct-DNA and 6 μM TO-3 with TO (A) or α-CN-TO (B). Samples were excited at 590 nm.
E
dx.doi.org/10.1021/ja308629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Figure 6. Confocal and fluorescence images of TO and α-CN-TO with HeLa cells. Samples were not washed before imaging. [Dye] = 1 μM.
and study of membrane receptor proteins involved in cystic
45−47
fibrosis.
Because α-CN-TO failed to bind to K7 or any other
previously identified scFv we tested, we decided to use the new
fluorogen as the bait to select new scFvs. As described
previously, the scFv library is displayed on the surface of yeast
9
25,48,49
cells and has a diversity of approximately 10 .
Mixing the
dye with the yeast-displayed library should result in a
fluorescent halo around those yeast that display scFv proteins
capable of binding and activating the dye. These winners can be
separated from the rest of the library by fluorescence activated
cell sorting (FACS). The individual yeast selected in this
manner are allowed to reproduce, and then the enriched library
is subjected to another FACS selection. The process is typically
repeated 4−6 times before individual yeast are grown as
isolated colonies. The gene corresponding to the selected scFv
can be isolated and sequenced and then used to express and
purify the scFv protein free of the yeast cell surface.
Figure 7. Crystal structures of TO (left) and α-CN-TO (right) at 0.83
Å resolution. The iodide counterion (magenta sphere) and acetonitrile
solvent molecule visible in the lower right image were omitted from
the upper right image for clarity. Note the proximity of the cyano
group to the benzothiazole sulfur (yellow) in the upper right image.
The selection process was performed to identify scFvs that
activate the fluorescence of α-CN-TO. Figure 8 shows confocal
fluorescence microscope images of yeast that display K7 or one
of the new scFvs, J3, stained with either TO-PRO-1 or α-CN-
TO. (TO-PRO-1 is a dicationic analogue of TO that was used
because the monocationic TO can enter cells and fluoresce.
26
TO-PRO-1 is known to bind to K7. ) A high degree of
selectivity is observed: K7 only activates TO, while J3 only
activates α-CN-TO. The origin of this selectivity cannot be
determined without high-resolution structural analysis of the
dye-protein complexes, but it is perhaps important that K7 was
selected for binding to a planar fluorogen, DIR, while J3 was
selected for binding to the nonplanar fluorogen α-CN-TO.
J3 was expressed and purified in soluble form. Binding of α-
CN-TO to purified J3 was studied by fluorescence spectros-
copy. The protein enhances the fluorescence of the dye
approximately 60-fold at 100nM each of protein and dye,
whereas TO exhibits almost no fluorescence (Figure 9)
consistent with the cell-surface imaging experiments (Figure 8).
Finally, we compared the photostability of the α-CN-TO/J3
and TO/K7 fluoromodules. As shown in Figure 10, the α-CN-
perhaps accounting for the lack of vibrational structure in the
UV−vis and fluorescence spectra shown in Figure 1.
Selection of an α-CN-TO-Binding Protein. In addition to
suppressing DNA binding, the nonplanar conformation of α-
CN-TO likely contributes to the failure of this dye to bind to
the highly promiscuous cyanine dye-binding protein K7 (Figure
S2 of the Supporting Information), which was originally
isolated from a library of single-chain antibody fragments
(
scFv) for binding to another fluorogenic dye, dimethylindole
26
red (DIR, Chart 1). These scFv-dye complexes, known as
fluoromodules, are finding increasing utility as alternatives to
other protein labeling technologies. Recent applications of
scFv-based fluoromodules include super-resolution imaging,
pH-sensitive tracking of endocytosis, protease biosensing,
42
43
44
F
dx.doi.org/10.1021/ja308629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Figure 8. Confocal fluorescence microscope images of yeast displaying
scFv proteins K7 or J3 at their surfaces and stained with 1 μM of either
TO-PRO-1 or α-CN-TO. Images were recorded without removing
excess unbound dye.
Figure 10. Photostability of α-CN-TO/J3 and TO/K7 fluoromodules.
Concentrations were adjusted to give equivalent initial absorbances at
the excitation wavelength of 470 ± 20 nm.
Nonplanar cyanine dyes were the subject of considerable
50
investigation by Brooker and co-workers (following earlier
51
work by Brunings and Corwin ), who reported that sym-
metrical cyanines that are forced to adopt a nonplanar
conformation exhibit red-shifted absorption spectra, while
strongly unsymmetrical (i.e., cases where the basicity of the
two heterocycles are significantly different) nonplanar cyanines
typically exhibit blue-shifted spectra. They also predicted that
nonplanar cyanines in which the heterocycles are different yet
have similar basicities, as is the case for benzothiazole and
5
2,53
quinoline,
would exhibit suppressed extinction coefficients
but no significant difference in absorption maxima. α-CN-TO
provides an example of such a compound, as its ε is nearly 50%
lower than TO with only a minor change in λmax. (The
electron-withdrawing nature of the CN substituent presumably
also helps to offset any red-shift that might arise from the
nonplanar conformation of the dye.)
Figure 9. Fluorescence spectra of α-CN-TO and TO in the presence
of purified scFv J3. [Dye] = [protein] = 500 nM. Samples were excited
at 488 nm.
The low nonspecific binding of α-CN-TO to DNA and
proteins and the absence of fluorescence when added to cell
culture makes the dye an excellent candidate for selection of
new scFv proteins from our yeast surface-displayed library. The
preliminary characterization of one such protein, J3, supported
our hypothesis that proteins could be found that would activate
α-CN-TO fluorescence and exhibit improved photostability.
The selection process that led to the isolation of J3 produced
several other α-CN-TO-binding proteins, which we are also
now characterizing.
TO/J3 fluoromodule exhibits superior photostability by at least
an order of magnitude.
DISCUSSION
■
The results presented above establish α-CN-TO as a new
fluorogenic cyanine dye with high photostability. Our initial
efforts to improve cyanine dye photostability relied on
fluorination of the dye heterocycles, but other studies
demonstrating that replacement of bridge hydrogens by
electron-withdrawing cyano groups leads to larger effects
motivated our synthesis of α-CN-TO. This dye shows
exceptional photostability and resistance to attack by singlet
oxygen, the main culprit in cyanine dye photobleaching.
The improvement in photostability upon α-cyano substitu-
tion of TO is much greater than what we observed previously
for the trimethine dye DIR, where the α-cyano group enhanced
photostability by a factor of 8, yet photobleaching of the dye
could still be readily observed in 90% glycerol in water. The
residual photobleaching of α-CN-DIR could be due to reaction
of singlet oxygen at other positions on the trimethine bridge, an
option that is not available in the monomethine dye α-CN-TO.
α-CN-TO is also potentially useful for selecting genetically
encodable RNA aptamers to monitor RNA expression and
localization, as reported for several other fluorogens, most
18
notably dyes modeled on the fluorescent component of GFP.
It should be possible to design and synthesize a family of
twisted cyanines that exhibit high photostability and cover a
fairly broad spectral range. We are currently in the process of
synthesizing and characterizing additional α-CN-substituted
monomethine cyanine dyes to broaden the catalogue of
photostable, fluorogenic cyanine dyes.
CONCLUSIONS
In the process of improving the photostability of thiazole
orange, a widely used fluorogenic cyanine dye, we discovered
■
G
dx.doi.org/10.1021/ja308629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
that addition of an electron-withdrawing cyano group to the
monomethine bridge of TO led to very high photostability,
virtually no reactivity toward singlet oxygen and elimination of
nonspecific DNA and protein binding leading to low
background fluorescence when added to mammalian cell
culture. We successfully selected a protein from a yeast-surface
display library that binds and activates the fluorescence of α-
CN-TO resulting in a new green fluoromodule with superior
photostability to previous TO-protein complexes. Ongoing
research is directed toward selecting and characterizing other α-
CN-TO-binding proteins and tuning the dye structure to
enhance brightness and extend the color palette of these new
photostable fluoromodules.
triethylamine the reaction mixture was allowed to stir
overnight. Roughly 3 volumes of water were slowly added to
the reaction flask with stirring during which time a
homogeneous solution was obtained followed by the
precipitation of the product. The solids were collected by
filtration and washed thoroughly with water. Yield 1.43 g (54%)
1
H NMR, (CDCl , δ ppm, 300 MHz): 3.31 (s, 3H), 4.21 (s,
3
1H), 6.90 (d, 1H, J = 8.1), 7.06 (t, 1H, J = 7.5 Hz), 7.27 (t, 1H,
J = 7.8 Hz), 7.38 (d, 1H, J = 7.8 Hz).
(
c). 4-Chloroquinoline (III). A neat solution of 5 g (34
EXPERIMENTAL SECTION
Reagents for the synthesis of α-CN-TO were purchased from
Alfa Aesar or Sigma Aldrich and used without further
purification. Solvents were HPLC grade. H NMR spectra
mmol) of 4-hydroxyquinoline and 16 mL (∼170 mmol) of
■
1
were recorded on a Bruker Avance 300 MHz spectrometer.
High-resolution mass spectra were acquired at the CUNY Mass
Spectrometry Facility at Hunter College on an Agilent
Technologies G6550 iFunnel Q-TOF mass spectrometer
equipped with Agilent’s Jet Stream electrospray ionization
source. Samples were introduced into the mass spectrometer
using an Agilent Technologies 1290 UHPLC system bypassing
the HPLC column. After dissolving in methanol, and diluting to
an appropriate concentration, the samples were injected at 200
ul/min using 90% methanol and 10% water containing 0.1%
formic acid. Settings for the ionization source were as follows;
drying gas temperature 225 °C, drying gas flow 17 l/min,
nebulizer pressure 20 psi, sheath gas temperature 200 °C,
sheath gas flow 12 l/min, Vcap 3500 V, nozzle voltage 500 V,
fragmentor 365 V. Profile and centroid mass spectral data were
collected over a range of 100−1700 m/z at 2 spectra per
second. The centroid data was used for the data analysis. The
instrument was operated in 2 GHz wide dynamic range mode.
POCl were heated to 110 °C for 2 h. The solution was allowed
3
to cool to room temperature and then slowly poured onto ice.
With cooling, solid sodium bicarbonate was used to neutralize
the solution and precipitate the product as a fluffy white solid.
The solid was filtered, washed with water, and then dried. Yield
1
4
.67 g (83%) H NMR, (CD CN, δ ppm, 300 MHz): 7.58 (d,
3
1
H, J = 4.7 Hz), 7.70 (t, 1H, J = 7.8 Hz), 7.81 (t, 1H, J = 7.8
Hz), 8.09 (d, 1H, J = 8.4 Hz), 8.23 (d, 1H, J = 8.4), 8.77 (d,
H, J = 4.7 Hz).
d). N-Methyl-4-chloroquinolium Iodide (IV). In a 15 mL
1
(
pressure reaction vessel was mixed 0.535 g (33 mmol) 4-
+
The reference masses used were purine with M+H at
chloroquinoline (III) and 2.33 g (102 mmol, 1.045 mL) of
methyl iodide. The tube was sealed and heated to 50 °C in an
oil bath. After the appearance of a precipitate, the reaction
mixture was heated for an additional hour and then allowed to
cool to room temperature. Once cooled, the yellow solids were
diluted further with ether and filtered. The obtained material
was an inseparable mixture of 4-chloro and 4-iodo derivatives in
a 1.6:1 ratio respectively. Yield (based upon 4-chloro) 0.941 g
+
1
21.050873 m/z and HP 922 with M+H at 922.009798 m/
z. They were infused into the spray chamber using Agilent’s
calibrant delivery system. The instrument was controlled with
Agilent MassHunter Workstation Acquisition Software version
B.05.00 and data was analyzed using Agilent MassHunter
Workstation Qualitative Analysis Software version B.05.00.
UV−vis and fluorescence spectra were recorded on Cary 3
and Cary Eclipse spectrophotometers respectively.
(
94%) 1H NMR, (DMSO-d , δ ppm, 300 MHz): mixture of
6
Synthesis of α-CN-TO. (a). 3-Methyl-2-(methylthio)-
benzothiazolium Iodide (I). In a 15 mL pressure tube was
chloro/iodo, 4.63 (s, 3H), 8.20 (t, 1H, J = 7.6), 8.39 (t, 1H, J =
.3), 8.52 (d, 1H, J = 6.5) 8.60 (m, 2H), 9.54 (d, 1H, 6.5).
e). 1-Methyl-4-[(3-methyl-2(3H)-benzothiazolylidene)-
8
(
cyanomethyl]-quinolinium Iodide (α-CN-TO). A homoge-
mixed 4.68 g (26 mmol) of 2-(methylthio)-benzothiazole and
1
0.93 g (77 mmol, 4.7 mL) of methyl iodide. The solution was
reacted at 100 °C at an upper power setting of 150 W for 15
min. The heavy white precipitate was filtered and washed with
ether. Yield 7.8 g (93%) H NMR, (DMSO, δ ppm, 300 MHz):
1
neous mixture was made from of 90 mg (0.5 mmol) 2-(3-
methyl-2(3H)-benzothiazolylidene)-acetonitrile (II) and 152
mg (0.5 mmol) 4-chloroquinolium iodide (IV) in a mortar and
pestle. The uniform solid was placed in a flask and heated to
150 °C for 30 min. The colored reaction was loaded directly
onto a silica column and the product, an orange-red fraction,
was eluted with increasing concentration of methanol (0−10%)
3
.13 (s, 3H), 4.11 (s, 3H), 7.72 (t, 1H, J = 7.9 Hz) 7.84 (t, 1H,
J = 8.1 Hz) 8.20 (d, 1H, J = 8.5 Hz), 8.41 (d, 1H, J = 8.1 Hz).
b). 2-(3-Methyl-2(3H)-benzothiazolylidene)-acetonitrile
II). A solution of 4.5 g (14 mmol) 3-methyl-2-(methylthio)-
(
(
benzothiazolium iodide (I) and cyanoacetic acid were dissolved
in 100 mL pyridine. After the addition of 1.41 g (14 mmol) of
H
dx.doi.org/10.1021/ja308629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
−
1
in methylene chloride. Yield 110 mg (51%) ε = 32,500 M
α-CN-TO. Colonies showing bright α-CN-TO fluorescence
were selected for further analysis.
−1
1
cm (491 nm, methanol), H NMR, (CD CN, δ ppm, 300
3
MHz): 3.47 (s, 3H), 4.33 (s, 3H), 7.45−7.64 (m, 4H), 7.80−
Fluorescence Microscopy. Frozen stock of yeast-surface
displayed J3 and K7 were inoculated into 5 mL of growth
medium and incubated at 30 °C. After two days, cells were
induced with galactose-containing induction media for three
days at 20 °C. Prior to imaging, an aliquot of 1:20 diluted
induced cells was washed three times with modified PBS buffer
followed by 10 min incubation with 1 μM α-CN-TO or TO-
PRO-1 on ice. Yeast cells were imaged without removal of
unbound dye using a Carl Zeiss LSM-510 META Confocal
Microscope. Samples were excited using a 488 nm laser and a
7
8
3
.87 (m, 2H), 8.09−8.20 (m, 2H), 8.54 (d, 1H, J = 8.5 Hz),
.59 (d, 1H, J = 6.7 Hz). ESI-MS (M+), 330.1061 m/z, calc.
30.1065 m/z
White Light Photobleaching. Samples were irradiated
with a 150 W Xe lamp using an Oriel lamp house (model
#6602) equipped with a water filter (Oriel #6117) and powered
by an Oriel 68805 universal power supply (40−200 W). The
white light was passed through a 380 nm glass long-pass filter.
Samples were immersed in an 18 ± 2 °C water bath during
irradiation periods.
Thermal Bleaching. Bleaching experiments in the presence
of 50 mM sodium molybdate and 9.8 mM hydrogen peroxide
contained 1 mM of dye. The buffer solutions were adjusted so
that the final ionic strength would be the same as the standard
wash buffer with pH 9.0. Hydrogen peroxide solution (10%)
was added to the buffer solution to give a final concentration of
5
05 nm long-pass filter. Images were processed using ImageJ
software. False color representative of the emission window was
applied.
Protein Purification. The pPNL6 plasmid containing J3
clone was extracted using Zymoprep I kit (Zymoresearch). The
R
plasmid was then transformed into Mach1-T1 E. coli.
Sequencing was performed by GeneWiz. Expression of the
25
soluble protein was accomplished as described previously.
9.8 mM. The absorbance and fluorescence spectra of the dye
and protein in the buffer solution were recorded, and then
ASSOCIATED CONTENT
Na MoO was added to the solution. Scans were taken at 10
■
2
4
min intervals over a total of 60 min.
*
S
Supporting Information
H NMR spectra for intermediates and α-CN-TO, time-lapse
1
Cell Culture Imaging. HeLa cells were grown, sub-
sequently centrifuged and resuspended in Dulbecco’s modified
eagle medium high glucose completed with 1% Penicillin/
Streptomycin, 0.025 M HEPES Buffer Solution, and 10% Fetal
Bovine Serum. 0.3 mL of the cell suspension and media were
added to Lab-Tek chambered #1.0 borosilicate coverglass 8
well plates and incubated for 24 h. After the incubation the
media was removed and replaced with 0.3 mL of a 1:1000
dilution of dye to media, which had been incubated until
reaching 37 °C. The cells were incubated with the dye for 20
min at 37 °C, after which the well plate was imaged on the
Olympus Fluoview 1000 Confocal (inverted microscope) using
a 60× oil 1.42NA objective. Dyes were excited with a 488 nm
laser and emissions were collected with a band-pass filter
between wavelengths 505−550 nm. The photomultiplier tube
levels were set to 660 V.
images of TO staining of HeLa cells, results of competition
assays for binding of TO and α-CN-TO to the single chain
antibody fragment protein K7, crystal structures of TO and α-
CN-TO in thermal ellipsoid representation, fluorescence and
DIC images for yeast-expressed scFv proteins stained with α-
CN-TO or TO-PRO-1, and amino acid sequence of J3 protein.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
*
Corresponding Author
Tel: 412-268-4196, fax: 412-268-1061, e-mail: army@cmu.
edu.
Notes
The authors declare no competing financial interest.
Selection of Yeast-Surface Display Clones That
Activate α-CN-TO. The naiv
used to select proteins that activate α-CN-TO consisted of ca. 8
̈
e yeast cell surface-display library
ACKNOWLEDGMENTS
■
8
×
10 recombinant human scFvs made from cDNA. The library
This work was supported by the U.S. National Institutes of
Health (grant U54 RR022241). NMR instrumentation at CMU
was partially supported by NSF (CHE-0130903). Mass
spectrometers were funded by NSF (DBI-9729351). We
thank Yehuda Creeger for assistance with FACS selections of
dye-binding proteins, Dr. Simon Watkins and Callen Wallace of
the University of Pittsburgh Center for Biologic Imaging for
performing the cell culture assays, and Dr. Steven Geib of the
University of Pittsburgh for crystallography services.
48,49
was developed by the Wittrup lab at MIT
and is also
available from Pacific Northwest National Laboratory. These
nonimmune scFvs were encoded in the pPNL6 plasmid as
fusion protein to the yeast cell surface protein Aga2p. EBY 100
yeast strain was used to host the display library.
Expression and enrichment of αCN-TO-activating scFvs
from the surface display library was performed as described
2
5
previously. Fluorescence Activated Cell Sorting (FACS) was
conducted on a Becton Dickinson FACSVantage SE. To
measure induction levels, induced cells were labeled with
AlexaFluor 647 goat antimouse antibody binding to a c-myc
epitope tag embedded in the scFv construct. Labeled cells were
then incubated with 1 μM α-CN-TO for an hour on ice prior to
sorting. A total of six rounds of enrichment and sorting were
conducted. Dye concentration was reduced after each round to
further enhance affinity. In the final selection round, a
concentration of 100 nM α-CN-TO was used to directly sort
clones onto an agar induction plate. After 3 days of growth at
REFERENCES
■
(1) Gonca
̧
lves, M. S. T. Chem. Rev. 2009, 109, 190−212.
(2) Suzuki, T.; Matsuzaki, T.; Hagiwara, H.; Aoki, T.; Takata, K. Acta
Histochem. Cytochem. 2007, 40, 131−137.
3) Vendrell, M.; Zhai, D.; Er, J. C.; Chang, Y.-T. Chem. Rev. 2012,
(
1
(
7
12, 4391−4420.
4) Wysocki, L. M.; Lavis, L. D. Curr. Opin. Chem. Biol. 2011, 15,
52−759.
5) Zhou, Y.; Xu, Z.; Yoon, J. Chem. Soc. Rev. 2011, 40, 2222−2235.
(6) Mishra, A.; Behera, R. K.; Behera, P. K.; Mishra, B. K.; Behera, G.
B. Chem. Rev. 2000, 100, 1973−2011.
(
20 °C, individual colonies were imaged in the presence of 1 μM
I
dx.doi.org/10.1021/ja308629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(
7) Ernst, L. A.; Gupta, R. K.; Mujumdar, R. B.; Waggoner, A. S.
Cytometry 1989, 10, 3−10.
8) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. T.; Waggoner, A. S.
Cytometry 1989, 10, 11−19.
9) Johnson, I.; Spence, M. T. Z. The Molecular Probes Handbook. A
(39) Hayashi, Y.; Shioi, S.; Togami, M.; Sakan, T. Chem. Lett. 1973,
651−654.
(
(40) Sovenyhazy, K. M.; Bordelon, J. A.; Petty, J. T. Nucleic Acids Res.
2003, 31, 2561−2569.
(
(41) Fei, X.; Gu, Y.; Lan, Y.; Shi, B.; Zhang, B. J. Chem. Crystallogr.
2011, 41, 1232−1236.
Guide to Fluorescent Probes and Labeling Technologies; 11th ed.;
Invitrogen/Molecular Probes, 2010.
(42) Fitzpatrick, J. A. J.; Yan, Q.; Sieber, J. J.; Dyba, M.; Schwarz, U.;
Szent-Gyorgyi, C.; Woolford, C. A.; Berget, P. B.; Waggoner, A. S.;
Bruchez, M. P. Bioconjugate Chem. 2009, 20, 1843−1847.
(43) Grover, A.; Schmidt, B. F.; Salter, R. D.; Watkins, S. C.;
Waggoner, A. S.; Bruchez, M. P. Angew. Chem., Int. Ed. 2012, 51,
4838−4842.
(
10) Benson, S. C.; Mathies, R. A.; Glazer, A. N. Nucleic Acids Res.
993, 21, 5720−5726.
11) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland,
R. P.; Mathies, R. A.; Glazer, A. N. Nucleic Acids Res. 1992, 20, 2803−
812.
1
(
2
(
(
12) Armitage, B. A. Top. Heterocyc. Chem. 2008, 14, 11−29.
13) Dash, S.; Panigrahi, M.; Baliyarsingh, S.; Behera, P. K.; Patel, S.;
(44) Falco, C. N.; Dykstra, K. M.; Yates, B. P.; Berget, P. B.
Biotechnol. J. 2009, 4, 1328−1336.
Mishra, B. K. Curr. Org. Chem. 2011, 15, 2673−2689.
14) Furutani, C.; Shinomiya, K.; Aoyama, Y.; Yamada, K.; Sando, S.
Mol. Biosys. 2010, 6, 1569−1571.
15) Sando, S.; Narita, A.; Aoyama, Y. ChemBioChem 2007, 8, 1795−
803.
16) Sando, S.; Narita, A.; Hayami, M.; Aoyama, Y. Chem. Commun.
008, 3858−3860.
17) Constantin, T.; Silva, G. L.; Robertson, K. L.; Hamilton, T. P.;
Fague, K. M.; Waggoner, A. S.; Armitage, B. A. Org. Lett. 2008, 10,
(45) Fisher, G. W.; Adler, S. A.; Fuhrman, M. H.; Waggoner, A. S.;
Bruchez, M. P.; Jarvik, J. W. J. Biomol. Screen. 2010, 15, 703−709.
(46) Holleran, J. P.; Brown, D.; Fuhrman, M. H.; Adler, S. A.; Fisher,
G. W.; Jarvik, J. W. Cytometry Part A 2010, 77A, 776−782.
(
(
1
(
2
(
47) Holleran, J. P.; Glover, M. L.; Peters, K. W.; Bertrand, C. A.;
Watkins, S. C.; Jarvik, J. W.; Frizzell, R. A. Mol. Med. 2012, 18, 685−
95.
48) Chao, G.; Lau, W. L.; Hackel, B. J.; Sazinsky, S. L.; Lippow, S.
M.; Wittrup, K. D. Nature Protocols 2006, 1, 755−768.
49) Feldhaus, M. J.; Siegel, R. W.; Opresko, L. K.; Coleman, J. R.;
5
(
(
(
1
(
(
561−1564.
Feldhaus, J. M.; Yeung, Y. A.; Cochran, J. R.; Heinzelman, P.; Colby,
D.; Swers, J.; Graff, C.; Wiley, H. S.; Wittrup, K. D. Nat. Biotechnol.
18) Paige, J. S.; Wu, K. Y.; Jaffrey, S. R. Science 2011, 333, 642−646.
19) Wilson, H. A.; Seligmann, B. E.; Chused, T. M. J. Cell. Physiol.
2
(
003, 21, 163−170.
50) Brooker, L. G. S.; White, F. L.; Sprague, R. H.; Dent, S. G.; Van
Zandt, G. Chem. Rev. 1947, 41, 325−351.
51) Brunings, K. J.; Corwin, A. H. J. Am. Chem. Soc. 1942, 64, 593−
00.
52) Brooker, L. G. S.; Keyes, G. H.; Williams, W. W. J. Am. Chem.
Soc. 1942, 64, 199−210.
53) Brooker, L. G. S.; Sklar, A. L.; Cressman, H. W. J.; Keyes, G. H.;
1
(
(
985, 125, 61−71.
20) Saltiel, J. J. Am. Chem. Soc. 1967, 89, 1036−1037.
21) Saltiel, J.; D’Agostino, J. T. J. Am. Chem. Soc. 1972, 94, 6445−
(
6
(
6
456.
(
22) Debler, E. W.; Kaufmann, G. F.; Meijler, M. M.; Heine, A.; Mee,
J. M.; Pljevaljcic, G.; Di Bilio, A. J.; Schultz, P. G.; Millar, D. P.; Janda,
K. D.; Wilson, I. A.; Gray, H. B.; Lerner, R. A. Science 2008, 319,
(
1
(
232−1235.
Smith, L. A.; Sprague, R. H.; Van Lare, E.; Van Zandt, G.; White, F. L.;
Williams, W. W. J. Am. Chem. Soc. 1945, 67, 1875−1889.
23) Simeonov, A.; Matsushita, M.; Juban, E. A.; Thompson, E. H.
Z.; Hoffman, T. Z.; Beuscher, A. E., IV; Taylor, M. J.; Wirsching, P.;
Rettig, W.; McCusker, J. K.; Stevens, R. C.; Millar, D. P.; Schultz, P.
G.; Lerner, R. A.; Janda, K. D. Science 2000, 290, 307−313.
(
24) Tian, F.; Debler, E. W.; Millar, D. P.; Deniz, A. A.; Wilson, I. A.;
Schultz, P. G. Angew. Chem., Int. Ed. 2006, 45, 7763−7765.
(
25) Szent-Gyorgyi, C.; Schmidt, B. F.; Creeger, Y.; Fisher, G. W.;
Zakel, K. L.; Adler, S.; Fitzpatrick, J. A.; Woolford, C. A.; Yan, Q.;
Vasilev, K. V.; Berget, P. B.; Bruchez, M. P.; Jarvik, J. W.; Waggoner, A.
Nat. Biotechnol. 2007, 26, 235−240.
̈
̈
26) Ozhalici-Unal, H.; Lee Pow, C.; Marks, S. A.; Jesper, L. D.; Silva,
(
G. L.; Shank, N. I.; Jones, E. W.; Burnette, J. M., III; Berget, P. B.;
Armitage, B. A. J. Am. Chem. Soc. 2008, 130, 12620−12621.
(
27) Shank, N. I.; Zanotti, K. J.; Lanni, F.; Berget, P. B.; Waggoner, A.
S.; Armitage, B. A. J. Am. Chem. Soc. 2009, 131, 12960−12969.
28) Zanotti, K. J.; Silva, G. L.; Creeger, Y.; Robertson, K. L.;
Waggoner, A. S.; Berget, P. B.; Armitage, B. A. Org. Biomol. Chem.
011, 9, 1012−1020.
29) Renikuntla, B. R.; Rose, H. C.; Eldo, J.; Waggoner, A. S.;
Armitage, B. A. Org. Lett. 2004, 6, 909−912.
30) Silva, G. L.; Ediz, V.; Armitage, B. A.; Yaron, D. J. Am. Chem.
Soc. 2007, 129, 5710−5718.
31) Toutchkine, A.; Nguyen, D.-V.; Hahn, K. M. Org. Lett. 2007, 9,
775−2777.
32) Doyle, F. P.; Kendall, J. D. Patent GB620802 (A), Great Britain,
949.
(
2
(
(
(
2
(
1
(
33) Lee, L. G.; Chen, C.; Liu, L. A. Cytometry 1986, 7, 508−517.
(
34) Ediz, V.; Lee, J.; Armitage, B. A.; Yaron, D. J. Phys. Chem. A
2
008, 112, 9692−9701.
35) Kutsche, I.; Gildehaus, G.; Schuler, D.; Schumpe, A. J. Chem.
Eng. Data 1984, 29, 286−287.
36) Wilkinson, F.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref.
(
(
Data 1993, 22, 113−262.
(
(
37) Aubry, J. M. J. Am. Chem. Soc. 1985, 107, 5844−5849.
38) Bohme, K.; Brauer, H.-D. Inorg. Chem. 1992, 31, 3468−3471.
̈
J
dx.doi.org/10.1021/ja308629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX