Rational Design and Facile Synthesis of a Highly Tunable Quinoline-Based Fluorescent Small-Molecule Scaffold for Live Cell Imaging

Article abstract of DOI:10.1021/jacs.8b03738

Small-molecule fluorescent probes are powerful tools for chemical biology; however, despite the large number of probes available, there is still a need for a simple fluorogenic scaffold, which allows for the rational design of molecules with predictable photophysical properties and is amenable to concise synthesis for high-throughput screening. Here, we introduce a highly modular quinoline-based probe containing three strategic domains that can be easily engineered and optimized for various applications. Such domains are allotted for (1) compound polarization, (2) tuning of photophysical properties, and (3) structural diversity. We successfully synthesized our probes in two steps from commercially available starting materials in overall yields of up to 95%. Facile probe synthesis was permitted by regioselective palladium-catalyzed cross-coupling, which enables combinatorial development of structurally diverse quinoline-based fluorophores. We have further applied our probes to live-cell imaging, utilizing their unique two-stage fluorescence response to intracellular pH. These studies provide a full demonstration of our strategy in rational design and stream-lined probe discovery to reveal the diverse potential of quinoline-based fluorescent compounds.

Full text of DOI:10.1021/jacs.8b03738

Article
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Rational Design and Facile Synthesis of a Highly Tunable Quinoline-
Based Fluorescent Small-Molecule Scaffold for Live Cell Imaging
Joomyung V. Jun, E. James Petersson,* and David M. Chenoweth*
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
S
* Supporting Information
ABSTRACT: Small-molecule fluorescent probes are powerful tools
for chemical biology; however, despite the large number of probes
available, there is still a need for a simple fluorogenic scaffold, which
allows for the rational design of molecules with predictable
photophysical properties and is amenable to concise synthesis for
high-throughput screening. Here, we introduce a highly modular
quinoline-based probe containing three strategic domains that can be
easily engineered and optimized for various applications. Such
domains are allotted for (1) compound polarization, (2) tuning of
photophysical properties, and (3) structural diversity. We successfully
synthesized our probes in two steps from commercially available
starting materials in overall yields of up to 95%. Facile probe synthesis
was permitted by regioselective palladium-catalyzed cross-coupling, which enables combinatorial development of structurally
diverse quinoline-based fluorophores. We have further applied our probes to live-cell imaging, utilizing their unique two-stage
fluorescence response to intracellular pH. These studies provide a full demonstration of our strategy in rational design and
stream-lined probe discovery to reveal the diverse potential of quinoline-based fluorescent compounds.
ologies, on fluorescent scaffolds, many rare and surprising
probes such as tricarbocyanine-based photostable near-infrared
(NIR) dyes8 or rosamine-based in vivo glutathione probes9
were developed.7 Yet, challenges still exist for both library
design and the ease of synthetic methods, stressing the
necessity of new tunable scaffolds that can be easily
diversified.6
Underexplored Quinoline Scaffold. Among many
synthetic scaffolds, quinoline is a highly attractive structure
for its relative synthetic versatility.4 Quinoline, being an
important construction motif, is widely observed in natural
products, drug discovery, as well as advanced functional
materials.4,10−12 Moreover, quinoline is the core structure of
quinine, a first well-defined small-molecule fluorophore,3,13 and
has been investigated as an intriguing molecular probe because
the nitrogen can be used as the site for monitoring the
interactions with target molecules through changes in
fluorescence.4,14,15 However, despite considerable efforts to
develop novel synthetic scaffolds containing heterocyclic rings,
quinoline has been underexplored as a fluorescent probe
largely due to synthetic difficulties.
INTRODUCTION
■
Fluorescence-based technologies have emerged as indispen-
sable tools for both in vitro and in vivo biomedical studies,
making them an essential facet of chemical biology.1−3
Fluorescent small molecules, in particular, have significant
advantages over protein-based probes in optical imaging and
analytical sensing due to their small, noninvasive size, high
sensitivity, and fast response time.2,3 Because they have
ubiquitous applications as cellular stains, environmental
sensors, or biomolecular labels, a plethora of fluorescent
probes have been developed over the past decades.3 Although
numerous fluorescent probes are known, the vast majority of
these probes are developed through structural modification of
a diminutive set of classical “core” dyes such as coumarin,
fluorescein, BODIPY, or cyanine.3 This underscores the
importance of intrinsic modularity of the “core” fluorophores
in the elaboration and modification of dyes, but also reveals the
limited number of available “core” scaffolds.3,4 Moreover,
within the context of biological systems, “ideal” fluorescent
probes are still in high demand, as properties such as cell
permeability, cytotoxicity, selectivity/sensitivity, and dynamic
range in the cellular environment are often difficult to predict
on the basis of rational design and theoretical calculations.5
Addressing these limitations, combinatorial approaches to
the discovery of fluorescent probes have emerged as exciting
alternatives for the exploration and optimization of fluo-
rophores.6,7 As a result of successful implementation of
combinatorial chemistry techniques, including major improve-
ments in high-throughput screening and imaging method-
Here, we introduce a small, yet highly tunable quinoline-
based “core” scaffold that is compatible with the generation of
libraries of fluorescent molecules in two steps (Figure 1). Our
scaffold includes three strategic domains that can be easily
functionalized and optimized to present potential advances
Received: April 6, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.8b03738
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Figure 1. (A) Modularity of dimethylamino quinoline (DMAQ) dyes consisting of a polarization domain and two tuning domains. (B) Two-step
synthesis of 2,4-disubstituted quinoline derivatives (15 monoarylated, 9 bisarylated DMAQ).
over classic and contemporary dyes. Multiwell plate analysis of
the resulting chemical libraries allowed us to identify useful
photophysical features in an efficient manner, and our
observations can serve as a guideline for next generation
library design based on rational structure/photophysical
relationships. We have further demonstrated the utility of
one derivative with particularly unusual pH-sensitivity in
multicolor live-cell imaging. We call this scaffold dimethyla-
mino quinoline or DMAQ.
yields. The initial DMAQ library was generated by (1) a one-
pot two-step cyclocondensation and chlorination procedure to
afford key precursor 3, and (2) a regioselective Suzuki−
Miyaura cross-coupling reaction at the 2-position (C-2) of
DMAQ (Figure 1B). The second DMAQ library was achieved
via a sequential cross-coupling reaction at C-2 and then at the
4-position (C-4).
Synthesis of the 2,4-Dichloroquinoline Core. The
synthesis of the key precursor of the DMAQ probe, 2,4-
dichloro-N,N-dimethylquinolin-7-amine 3, was first attempted
following previous reports, which involved two steps,
inefficient purifications such as filtration or recrystallization,
and harsh conditions such as extremely high temperatures, up
to 270 °C.29,30 Adapting the reported conditions, 3-
(dimethylamino) aniline 1 was first cyclocondensed with
neat diethyl malonate to form 7-(dimethylamino)-4-hydrox-
yquinolin-2(1H)-one 2. After filtration of solids, 2 was
chlorinated using neat phosphorus oxychloride (POCl3)
under reflux conditions to afford dichloroquinoline 3. The
previously reported yield for compound 3 was estimated to be
54% (Figure S2);29,30 however, we found that these conditions
resulted in only 26% yield on the gram scale (Table 1, entry 1),
which is similar to the 25% yield of 2,4-dibromo-N,N-
dimehtylquinolin-7-amine in a recent report (Figure S2).30
RESULTS AND DISCUSSION
■
Rational Design of DMAQ Scaffold. In general, a simple
and short synthetic route to generate a large number of target
molecules is favored.16,17 However, most commonly used dyes
require several steps for derivatization, resulting in low overall
yields because they lack easy tunability or late-stage
modification sites.6 To achieve desired step economy in
obtaining dyes of interest, a highly efficient synthetic route to
obtain a key precursor and its derivatives was investigated.
Therefore, we designed our scaffold to contain three strategic
domains: one for polarization and two tunable domains for
structural diversity and/or late-stage chemical modification
(Figure 1A). Polarization of the fluorophore is achieved by the
electron-donating dimethylamino group at the 7-position of
quinoline, which can push electron density into the core of
DMAQ. The 2- and 4-positions of DMAQ are allotted for
tuning domains, where electronically and structurally diverse
chemical moieties can be installed to either push or pull the
electron density. These substitutions were envisioned to
orchestrate a versatile “push−pull” system of the fluorescent
scaffold (Figure 1A). Generated molecules with a D(donor)-π-
A(acceptor) system are anticipated to display varied photo-
physical (e.g., fluorescence, quantum yield, solvatochromism,
Stokes shifts, etc.) and chemical properties (e.g., solubility, ion
binding, acidity/basicity, etc.).
a
Table 1. Conditions for Synthesis of Key Precursor 3
entry
solvent
T (°C)
time (h)
POCl3 (equiv)
yield (%)
1
2
3
4
5
none
DPE
DPE
DPE
DPE
180−270
160−200
160−200
160−190
160
5
15
15
>20
>24
6
10
10
10
3
<26
96
72
80
75
b
To accelerate the utilization of our scaffold, a synthetic route
for rapid assembly of the DMAQ core was studied. Not
surprisingly, over the past decades, several condensation and
cascade reactions to efficiently access medicinally valuable
multisubstituted quinolines have been reported with various
mechanisms and conditions, including Conard−Limpach−
Knorr,18,19 Skraub−Doebner−Von Miller,20−22 Fried-
laender,23,24 and halogen25 or copper26 mediated.4,27,28
However, many traditional condensation methods suffer from
low yields, harsh reaction conditions, and operational
complications, or require specialized starting materials or
catalysts.4,28 In this work, we report a significantly improved
two-step synthesis of quinoline-based dyes from commercially
available starting materials, which provides good to excellent
a
All reactions in the table above were performed on the same scale
(8.8 mmol) and monitored by thin layer chromatography (TLC)
b
analysis for formation of 2. Reported yield was obtained after
intermediate 2 was isolated. DPE is diphenyl ether.
The poor reproducibility and low yield that we observed was
presumed to be due to heterogeneity during the cyclo-
condensation reaction. Moreover, poor solubility of hydrox-
yquinolone 2 made column purification difficult and further
affected the yield in the following halogenation reaction. We
envisioned that an appropriate solvent with a high boiling
B
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point would keep 2 in solution and ensure that the reaction
mixture remained homogeneous. Gratifyingly, we found that
diphenyl ether (DPE), which has boiling point of 258 °C, not
only keeps the reaction mixture in a homogeneous state, but
also is inert to both cyclocondensation and halogenation
reaction conditions, allowing one-pot two-step synthesis of 2 in
an excellent yield on gram scale. Complete formation of 2 was
monitored via thin layer chromatography (TLC) analysis
(Figure S3B), which was then followed by addition of POCl3
to obtain 3 in 96% overall yield (Table 1, entry 2). The yield
dropped to 72% when the chlorination reaction was performed
after intermediate 2 was isolated by solid filtration (Table 1,
entry 3). Cyclocondensation reactions were also attempted in
milder conditions such as lower temperature (190 or 160 °C)
or fewer equivalents of POCl3. Those conditions successfully
afforded 2, but resulted in lower conversion and longer
reaction times than the entry 2 conditions (Table 1, entries 4
and 5). Thus, our one-pot, optimized conditions efficiently
produce poly halogenated quinoline 3, a core subunit that can
be utilized in transition-metal-catalyzed cross-coupling reac-
tions to generate our fluorogenic probes as well as medicinally
valuable bioactive compounds.31
the basis of the previous report that showed high C-2
selectivity for dichloropyridine.16 Using 0.6 mg (2.5 μmol) of
dichloroquinoline 3 per reaction, a total of 96 conditions were
screened (Figure S4). Not surprisingly, mixtures of mono-
coupling products (4, 4′), bis-coupling product (5),
dechlorinated byproduct, and hydrolyzed byproducts were
observed from these reactions (Tables S1−4 and Figures S5−
7). As summarized in Table 2, the ligands exert a profound
Table 2. Selected Results of Regioselective Cross-Coupling
a
Reactions with Three Electronically Distinct Boronic Acids
Synthesis of Monoarylated DMAQ via Regioselective
Cross-Coupling Reaction. Palladium-catalyzed cross-cou-
pling reactions of halogenated heteroarenes have played a
pivotal role in the synthesis of important biological targets or
their key precursors.32 We were interested in identifying a
general catalyst system capable of C-2 selective arylation of
dichloroquinoline 3, which would give us the flexibility for
structure−photophysical property relationship studies of
fluorophores. However, with heteroarenes bearing identical
halogens, high selectivity is difficult to achieve as the final
regioselectivity is determined by multiple factors including
steric effects, electronic effects, and the presence of directing
groups in the reactants.32 While many conditions have been
investigated to achieve C-2 selective functionalization of 2,4-
dichloroquinoline or 2,4-dichloropyridine, there was no
reported condition for 3, which includes an electron-donating
substituent at the 7-position.32,33 Unlike 2,4-dichloroquinoline,
which possess only one highly electrophilic position at C-2, 3
contains the dimethylamino group that could reduce the
electrophilicity of C-2 and weaken selectivity. Additionally, the
C-2 selectivity of the pyridine moiety seems to be highly
dependent on the choice of ligand or the presence of additives
such as LiCl, which could alter the selectivity to C-4.32,33
Finally, consideration must be given to avoid bis-arylated
products, as we would like to keep C-4 as an additional site for
late-stage structural diversification or functionalization. In an
effort to achieve highly regioselective and broadly compatible
catalyst condition for electronically diverse aryl boronic acids,
we screened several factors: electronically distinct boronic
acids, ligands, bases, solvents, and temperature. With the
support of the Penn/Merck high-throughput experimentation
center, a general and efficient condition for C-2 selective
Suzuki−Miyaura cross-coupling of 3 was investigated.
a
Four selected catalyst conditions are shown for each boronic acid:
triphenylphosphine (PPh3), 1,3-bis(diphenylphosphino) propane
(DPPP), 1,1′-bis(di-isopropylphosphino)ferrocene (DIPPF), and
tricyclohexylphosphine (PCy3). Entry 2, 6, and 9 are the best
conditions for the corresponding boronic acids. The final solvent
consists of 4:1 solvent:water. Percent composition of products was
determined by chromatographic peak integration normalized with an
internal standard.
effect on the regioselectivity of the coupling as well as optimal
reactivity to minimize bis product.32 Unlike previously
reported conditions for C-2 selective Suzuki−Miyaura cross-
coupling of dichloropyridine using 1,1′-ferrocenediyl-bis-
(diphenylphosphine) (DPPF), it was observed that, in general,
triphenylphosphine (PPh3) and 1,3-bis(diphenylphosphino)
propane (DPPP) highly favored coupling at C-2 over C-4
(Table 2, entries 1, 2, 5, 6, 9, and 10), while 1,1′-bis(di-
isopropylphosphino)ferrocene (DIPPF) and tricyclohexyl-
phosphine (PCy3) gave a low yield for 4 (Table 2, entries 3,
4, 7, 8, 11, and 12) due to poor C-2 selectivity or a high yield
of 5. Nonetheless, our desired 4 was the major product in most
screened conditions, presumably due to the intrinsic reactivity
of CN bond and the effect of the quinoline nitrogen
coordinating to the Pd catalyst, confirming that the order of
the relative ease of oxidative addition for 2,4-dichlorquinoline
is C-2 > C-4.32−34
After additional temperature screening at 60, 70, and 80 °C,
we established that performing the reaction with 5 mol % of
bis(triphenylphosphine) palladium chloride (Pd(PPh3)2Cl2)
and 3 equiv of K2CO3 in 4:1 tetrahydrofuran (THF):water at
65 °C for 5−48 h afforded products in the highest yield with
excellent C-2 regioselectivity for a broad scope of boronic
acids. A range of aryl boronic acids, spanning electron-donating
to -withdrawing para-substituted phenylboronic acids, steri-
Screening was conducted with 5 mol % palladium acetate
(Pd(OAc)2), 5 mol % (bidentate) or 10 mol % (monodentate)
of eight different ligands, two different bases, cesium carbonate
(Cs2CO3) or potassium carbonate (K2CO3) (3.0 equiv), and
three different phenylboronic acids (1.1 equiv). We chose 4-
(dimethylamino)phenyl-, phenyl-, and 4-(cyano)phenyl-bor-
onic acids as three representative electron-rich, -neutral, and
-deficient substrates, respectively. Eight ligands were chosen on
C
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cally hindered arylboronic acids, and heteroaryl boronic acids,
were subjected to the optimized reaction condition (Scheme
1). All of the electron-rich (4b), electron-neutral (4c,d), and
both medicinal chemistry and organic material design,37 and a
primary amine is a convenient site for conjugation for
biological studies. Interestingly, electron-withdrawing aryl
boronic acids reacted much more rapidly with starting material
3 than did electron-donating boronic acids. The overall scope
of the study showed the robustness and generality of the
reaction condition to electronic differences, steric hindrance,
and varied functional groups with high yields and excellent
regio-selectivity. This enabled the rapid construction of diverse
arrays of monoaryl- or heteroarylquinolines.
a
Scheme 1. Aryl/Heteroaryl Boronic Acid Substrate Scope
Effect of the C-2 Substituents on Photophysical
Properties of DMAQ. Having accomplished the facile
synthesis of a DMAQ library, we then turned our attention
to studying the photophysical properties generated by
variations in the push−pull system of DMAQ. Upon initial
visual inspection, more electron-withdrawing (hetero)aryl
substitution on C-2 of DMAQ showed more red-shifted
emission in DMSO, indicating the formation of a D-π-A
system (Figure 2A). To provide a guideline for rational
a
All reactions performed on 0.18 mmol scale. Reported yields are
isolated yield.
electron-poor (4e,f) phenylboronic acids afforded excellent
isolated yields (>91%) of C-2 arylated products. Of sterically
hindered phenylboronic acids, the presence of a methoxy
functional group at the ortho- and para-positions resulted in
92% (4g) and 94% (4i) yields, respectively, while at the meta-
position it gave a slightly lower isolated yield of 83% (4h).
Although phenyl-substituted 4a resulted in a relatively low
yield35 (39%), high yield for para-methoxy phenyl-substituted
4i demonstrates protected phenol can be utilized for synthesis
of 4a. To assess additional steric effects, 2,4-difluorophenylbor-
onic acid and 9-anthraceneboronic acid were subjected to
reaction with 3. While 4j was afforded in 87% yield, only a 20%
yield was observed for 4k, although it was still the major
arylated product. The low yield of 4k is most likely due to
protodeboronation of the anthracene boronic acid, as
significant amounts of anthracene product were observed
(Figure S8).36 Furthermore, we examined C-2 arylation with
heterocycles, 2-thienyl and 3-thienyl boronic acid, to afford 4m
and 4n in 85% and 97% yields, respectively. The cross-
coupling condition tolerated a variety of para-substituted
functional groups, such as hydroxyl, methoxy, cyano,
dimethylamino, and fluoro as well as a Boc-protected primary
amine. Fluorine and primary amines are particularly important
functional groups as fluorine is a frequently used substituent in
Figure 2. Structure−photophysical property relationship of DMAQ.
(A) A range of C-2 aryl substitution with emission maximum under
405 nm excitation is shown. The compound order is 4b, 4a, 4n, 4i, 4c,
4d, 4m, 4l, 4g, 4h, 4j, 4e, 4f, 4k, and 4l′ from left to right. Stock
solutions of 4a−4n were made in DMSO (5 mM) and 4l′ in water
with 0.1% TFA (5 mM). The image was taken using a hand-held UV
lamp (365 nm). (B) Correlationbetween emission wavelength and
Hammett constant (σp) of R1 substituents in monoaryl DMAQ (R2 =
0.79).
selection of substituents for future DMAQ derivatives, we
analyzed 4b, 4a, 4i, 4c, 4d, 4e, and 4f in terms of the Hammett
substituent constant for the para functional group on the C-2
phenyl ring (Figure 2B).38 Electronegativity and emission
wavelength showed a positive correlation, as expected. This
analysis is further supported by HOMO−LUMO calculations
that show a movement of electron density from the C-7
dimethylamino group to the C-2 aryl group upon excitation
from S0 to S1 (see full discussion in Tables S15,S16 and
Figures S31−S36).
Photophysical Properties of Monoaryl DMAQ. Strat-
egies in fluorescent probe development consider not only the
design and synthesis of fluorescent libraries, but also the
evaluation and validation of the probes in a high-throughput
D
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Figure 3. Plate reader analysis of photophysical properties of monoaryl DMAQ. Each compound (3,4a−4n) was prepared as a 5 mM stock solution
in diemthyl sulfoxide (DMSO) except for 4l′ in water. Each well contains 1% DMSO in solution with a final 50 μM dye concentration. For polarity
screening (left panel), toluene, basic alumina filtered dichloromethane (DCM), 0.1% trifluoroacetic acid in DCM (DCM+TFA),
dimethylformamide (DMF), DMSO, and ethanol (EtOH) were used as solvents. For pH screening (right panel), 10 mM buffer solutions were
prepared; pH 2 (hydrochloric acid), pH 4 (citric acid/phosphate buffer), pH 6.9 (citric acid/phosphate buffer), pH 7.3 (phosphate buffer), pH 10
(carbonate buffer), and pH 11 (sodium hydroxide). Emission spectra were measured at λex of 405 nm regardless of their optimal max absorption,
and the image was taken under a hand-held UV lamp (365 nm).
manner. Thus, to obtain more quantitative information on the
properties of our DMAQ library, we designed a multiwell assay
amenable to assessing spectral properties such as fluorescence
intensity and emission wavelength for each compound in the
library (Figure 3).
filtered DCM (Figure 3B). Under nonpolar protic/acidic
conditions (DCM+TFA), all compounds 4a−4n showed a
significant bathochromic emission shift in comparison to
emission in basic alumina-filtered DCM (Figure 3A). While 3
had a minimal bathochromic shift of 3 nm, (hetero)arylated
DMAQs (4b, 4c, 4e, 4f, 4i, 4l, 4m) showed an 84−96 nm shift
in the presence of 0.1% TFA. These results indicate the general
pH sensitivity of the quinoline “core” scaffold. Moreover, many
dyes showed a sensitivity to solvent polarity as well as pH. 4k,
in particular, was highly solvatochromic, with an emission
spanning 480−586 nm in various organic solvents (Figure 3C).
This can be a very valubale property in which the dye could be
used to sense its local environment on a protein surface or in a
cellular compartment. Boc deprotection of 4l gave 4l′ with
significantly increased water solubility and enhanced brightness
(Figure 3D).
Dyes with explicitly pH-sensitive functional groups were also
investigated, where the emission of phenol-functionalized 4a
red-shifted in acidic environments, with an apparent pKa in the
4−6.9 range (Figure 3E). The pH range for fluorescence
changes can be tuned through electronic effects; fluorine-
functionalized 4d exhibited a 118 nm red-shift in emission on
going from pH 4 to pH 2 (Figure 3G), while the emission of
ortho-methoxy-functionalized 4g red-shifted 100 nm on going
from pH 7 to pH 4 (Figure 3H). The fluorescence of
dimethylamino-substituted 4b showed an interesting three-
stage change with pH, where it was quenched at acidic pH’s,
and had two emission peaks at 480 and 600 nm at pH 7, with
the second peak growing more prominent at basic pH’s
The emission spectrum of each compound was measured
under excitation at 405 nm, a common wavelength laser used
in confocal microscopes for biological studies. Because the
dimethylamino group and quinoline nitrogen in the DMAQ
derivatives were expected to induce solvatochromism and pH
sensitivity, six organic solvents with varying properties were
chosen: nonpolar aprotic (toluene, DCM), nonpolar protic/
acidic (DCM+TFA), polar aprotic (DMF, DMSO), and polar
protic (EtOH). In addition, six different buffered aqueous
solutions spanning pH 2 to pH 11 were used for screening.
While our parent compound 3 showed no significant
environmental sensitivity, the rest (4a−4n) of the 2,4-
substituted quinoline dyes showed solvent-dependent photo-
physical properties, confirming the design principle of
tunability (spectra selected to illustrate certain observations
are shown in Figure 3; complete sets of emission spectra for all
dyes are shown in Figures S9 and 10). Furthermore, this
tunability was generally predictable, with electron-withdrawing
para-substituted phenyl rings at the 2-position red-shifting the
emission (e.g., 4f) as one would expect for the designed push−
pull system.
Selected DMAQs with para-substituded aryl rings (4b, 4c,
4e, 4f, 4i, 4l), (hetero)aryl rings (4m), and parent core DMAQ
3 showed emission spanning 453−516 nm in basic alumina
E
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(Figure 3F). Taken together, these data show the importance
of the C-2 position in tuning the spectral properties of DMAQ
dyes. The monoaryl DMAQ derivatives can easily be
functionalized at C-4 for conjugation to other molecules or
for further derivatization.
Synthesis of Bisaryl DMAQ via Sequential Cross-
Coupling Reaction. The ability to regioselectively cross-
couple aryl groups at the C-2 position of 3 provided us with
the opportunity to further tune the properties of our dyes by
substitution at the C-4 position. In screening conditions for
sequential cross-coupling, we found that once the 2-arylated
product was formed using our optimized regioselective cross-
coupling conditions, a second arylation cross-coupling reaction
could be performed without additional Pd by addition of an
excess of a second boronic acid and increasing the temperature
to 85 °C (Figure 4A). Via the three boronic acids used in
strongly blue-shifted to 483 nm by addition of an electron-
donating amino group on C-4 to give 5g (Figure 4D).
Interestingly, 5c and 5g both had the same set of substitutions,
but in opposite positions, giving very different properties: 5c
exhibited highly red-shifted emission, while 5g was highly blue-
shifted. This phenomenon implies that excitation involves the
flow of electrons from the C-2 substituent to the C-4
substituent, so that matching this with appropriate electron-
donating (C-2) and electron-withdrawing (C-4) groups lowers
the energy for this transition and red-shifts the absorption
(Table S5) and emission.
Photophysical Property of Bisaryl DMAQ. 5a−i were
subjected to pH-dependent fluorescence spectral analysis by
screening their emission in pH 2, 4, 6.9, 7.3, 10, and 11 as
shown in Figure 3 for 4a−4n (Figure S14). 5a, 5b, 5d, 5e, and
5f exhibited pH-dependent dual emissive properties, where
emission red-shifted up to 170 nm in solutions of pH 2 and/or
pH 4 under single excitation at 405 nm. 5g and 5i showed pH-
dependent intensity changes; 5g had a 20-fold higher emission
at 525 nm in pH 11 buffer than in pH 2 buffer. Last, 5c had
quenched emission in buffers at or below pH 4, while 5h had
quenched emission at pH 2 (Figures S20 and S21) under
excitation at 405 nm.
Live Cell Imaging of DMAQ Dyes. The DMAQ
compounds (4a−4n, 5a−5i) were all subjected to live HeLa
cell imaging (Figures S15−17). All of them, except for 4f, were
cell permeable and were suitable for live cell imaging. Yet it
should be noted that modifying 4f to 5g−5i enabled good cell
permeability, validating the benefit of tunable scaffold. Selected
dyes were subjected to cytotoxicity assays (Table S6). Among
them, an application of pH-sensitive dye 5a in live cells was
chosen as a proof-of-concept to illustrate the value of DMAQs.
5a demonstrated superior properties over many commercial
pH sensitive probes (Figure 5). In a neutral to basic
environment (pH > 7), a broad absorption band with λex at
308 and 412 nm (green dotted line) and λem at 469 nm (green
solid line) of 5a was observed (Figure 5A). However, at a pH
of 4 or lower, absorbance and emission shifted to λex at 474 nm
(red dotted line) and λem at 637 nm (red solid line),
respectively. Having a bathochromic shift of 168 nm in
emission from the basic to acidic environment produced a
large Stokes shift and gave 5a an unusual pH-dependent dual-
emission characteristic. These properties allowed 5a to stain
two different locations based on live HeLa cells. Green staining
(Figure 5C) obtained by exciting at 405 nm and collecting the
emission at 475 25 nm (Figure 5C) identified a subset of
probe molecules presumably sequestered in neutral or basic
compartments. Red staining was produced by exciting at 488
nm with the emission filter set at 650 25 nm (Figure 5D)
and indicated a substantial fraction of the dye in a presumably
acidic environment. A merged image did not show any yellow
regions, indicating that 5a can orthogonally image two regions
that differ in local environment (Figure 5F). This is in contrast
to many pH-sensitive dyes, which are based on a change in
fluorescence quenching. For instance, the commercial dye, cell-
permeant ratiometric pH indicator seminaphthorhodafluor
(SNARF) exhibits a pH-dependent emission shift from acidic
to basic conditions. This pH dependence allows the ratio of
the fluorescence intensity from the dye at two emission
wavelengths, typically 580 and 640 nm, to be widely used for
quantitative determination of pH. However, SNARF deriva-
tives are only sensitive to pH changes near 7 and have a
relatively small (60 nm) emission shift with changes in pH.
Figure 4. Sequential cross-coupling for bisaryl DMAQ dyes. (A)
Reaction scheme for sequential cross-coupling. (B−D) Emission
spectra of 5a−5i in DMSO upon 405 nm excitation. They are
categorized under the same R1 where (B) R1 = N(CH3)2, (C) R1 = H,
and (D) R1 = CN. Reported yield is isolated yield.
Table 2, we investigated the efficiency of sequential cross-
coupling. To run sequential coupling reactions at 85 °C,
dioxane was used instead of THF. Gratifyingly, we found that
all reaction sequences gave good to excellent yields (HPLC
yields shown in Figures S11−13).
Effects of the C-4 Substituents on Photophysical
Properties of DMAQ. The nine-compound doubly subsituted
DMAQ library was used to study the effect of the C-4 domain
on DMAQ photophysical properties. Generally, electron-
withdrawing groups on the C-4 phenyl ring red-shifted the
emission of the corresponding parent DMAQ 4b, 4c, or 4f. 5c
was particularly strongly affected by para-cyanophenyl
modification at C-4, resulting in a highly red-shifted emission
maximum of 593 nm as compared to 482 nm for 4b (Figure
4B). Conversely, the emission maximum of 4f (576 nm) was
F
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Journal of the American Chemical Society
Article
DMAQ could be rationally tuned by C-2 substituents, and
DMAQ dyes generally exhibited a large Stokes shift. Our study
of 2,4-diarylated DMAQs revealed a probable photophysical
mechanism for electron flow from the C-2 substituent to the
C-4 substituent, which can be explored in subsequent
theoretical and experimental investigations. Building off of
our results with 5c, further accentuating the electron donation
of the C-2 position and electron withdrawal of the C-4 position
could generate very small near IR dyes. Studies of the cellular
localization of 5a and other dyes presented here will allow the
microscopy community to take advantage of their unique
properties. On the basis of these promising initial inves-
tigations, we expect to develop DMAQ as a multifunctional
probe that can serve as both drug and sensor to answer
challenging questions in complex biological processes. We
envision that highly tunable and optimizable fluorescent
probes can function in convenient, continuous assays, thereby
providing useful tools for studying biological events in cellular
environments.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b03738.
■
S
Figure 5. Fluorescence spectrum and live HeLa cell imaging after 3 h
incubation of 5a. (A) Normalized UV absorption spectra and
emission spectra excited by 405 nm are shown in pH 7 and pH 2
buffer. 405 and 488 nm lasers for microscopy excitation are shown by
arrows. (B) Differential interference contrast (DIC) image of Hela
cells after 3 h incubation of 5a. (C) λex = 405 nm, λem = 425−475 nm.
(D) λex = 488 nm, λem = 650−700 nm. (E−G) Merged images of
green (λex = 405 nm, λem = 425−475 nm) and red (λex = 488 nm, λem
= 625−675 nm) in (E), red (λex = 488 nm, λem = 650−700 nm) in
(F), and red (λex = 488 nm, λem = 700−750 nm) in (G).
Experimental protocols, characterization data, and NMR
spectra of all new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*ejpetersson@sas.upenn.edu
*dcheno@sas.upenn.edu
ORCID
David M. Chenoweth: 0000-0002-0819-4669
Notes
Moreover, the published syntheses of SNARF variants are
challenging (2−7 steps and <18% yield).39−41 These
limitations are addressed by 5a, which exhibits a 170 nm
pH-dependent emission shift, and other DMAQ derivatives, all
of which are easily synthesized, with a tunable pH sensor range
that can be seen in Figure S14.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by funding from the University of
Pennsylvania, by the National Science Foundation (NSF CHE-
1708759 to E.J.P.), and by the National Institutes of Health
(Grant NIH R01-GM118510 to D.M.C.). Instrument support
from the National Institutes of Health (Grant NIH RR-
023444) includes HRMS. We thank Dr. Charles W. Ross III
(University of Pennsylvania) for assistance in HRMS analysis,
Dr. Patrick Carroll (University of Pennsylvania) for X-ray
analysis, Dr. Jun Gu (University of Pennsylvania) for NMR
assistance, and Dr. Simon Berritt (University of Pennsylvania)
for helpful discussion of high-throughput experimentation.
CONCLUSIONS
■
Our work demonstrates a synthetic advance to develop the
underexplored quinoline as a fluorescent scaffold “core” that
allows both rational and combinatorial approach to create
novel dyes. Our method has also served to simplify the
synthesis of medicinally important substituted quinoline
analogues. The power of the rationally designed DMAQ
“core” scaffold in the discovery of new DMAQ dyes has been
validated, although by no means fully explored. There is an
exciting and immediate opportunity for the exploration and
optimization of DMAQ probes for a wide range of
applications. For instance, these DMAQ fluorophores exhibit
solid-state fluorescence in amorphous and/or crystalline solids,
which have potential for application in organic materials
(Figure S22). Combining a rationally designed tunable scaffold
with high-throughput analysis, we show more than 20 different
2,4-disubstituted DMAQ derivatives with a range of emission
spectra spanning the visible region as well as interesting
chemical properties. With this library, we were able to further
understand the structure−photophysical relationship and
intrinsic properties of the DMAQ scaffold. Emission of
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