Quinoline-based fluorescent small molecules for live cell imaging

Article abstract of DOI:10.1016/bs.mie.2020.04.060

Small molecule probes are essential tools for biomedical applications, with utility as cellular stains, labels for biomolecules, environmental indicators, and biosensors. However, a fluorophore's characteristics are difficult to predict solely through calculations or rational design, making the development of a core scaffold that is amenable to late stage functionalization particularly desirable. In this chapter, we describe the synthesis and application of a tunable quinoline scaffold that can be readily functionalized and optimized for a variety of imaging applications. We present a facile synthesis that results in three functional domains that influence the compound's photophysical properties, structural diversity, and polarization. We demonstrate a method with which to study the scaffold's tunable photophysical properties as a result of its structure and environment, and finally exhibit its utility in pH sensitive, live-cell imaging.

Full text of DOI:10.1016/bs.mie.2020.04.060

CHAPTER FIFTEEN
Quinoline-based fluorescent small
molecules for live cell imaging
Rachel M. Lackner, Joomyung V. Jun, E. James Petersson∗,
David M. Chenoweth*
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia,
PA, United States
*Corresponding authors: e-mail address: ejpetersson@sas.upenn.edu; dcheno@sas.upenn.edu
Contents
1. Introduction
310
310
311
313
313
314
316
317
319
320
320
321
322
323
323
324
324
325
1.1 Small molecule fluorophores
1.2 Environmentally sensitive dyes
2. Chemical synthesis
2.1 Reagents, equipment, and general procedures
2.2 Synthesis of 2,4-dichloroquinoline core
2.3 Synthesis of monoarylated quinolines, 4a-n
2.4 Synthesis of bisarylated quinolines, 5a-i
2.5 Synthetic notes
3. Plate reader analysis of DMAQ derivatives
3.1 Reagents and equipment
3.2 Procedure
4. Cell imaging of an environmentally sensitive dye
4.1 Reagents and equipment
4.2 Procedure
4.3 Analysis
5. Summary and conclusions
References
Abstract
Small molecule probes are essential tools for biomedical applications, with utility as cel-
lular stains, labels for biomolecules, environmental indicators, and biosensors. However,
a fluorophore’s characteristics are difficult to predict solely through calculations or ratio-
nal design, making the development of a core scaffold that is amenable to late stage
functionalization particularly desirable. In this chapter, we describe the synthesis and
application of a tunable quinoline scaffold that can be readily functionalized and
#
Methods in Enzymology, Volume 640
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2020.04.060
2020 Elsevier Inc.
All rights reserved.
309

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Rachel M. Lackner et al.
optimized for a variety of imaging applications. We present a facile synthesis that results
in three functional domains that influence the compound’s photophysical properties,
structural diversity, and polarization. We demonstrate a method with which to study
the scaffold’s tunable photophysical properties as a result of its structure and environ-
ment, and finally exhibit its utility in pH sensitive, live-cell imaging.
Abbreviations
DCM
DMAQ
DMF
dichloromethane
dimethylamino quinoline
dimethylformamide
DMSO
EtOAc
EtOH
HPLC
HRMS
K2CO3
LCMS
MeOH
NMR
POCl3
TFA
dimethyl sulfoxide
ethyl acetate
ethanol
high performance liquid chromatography
high resolution mass spectroscopy
potassium carbonate
liquid chromatography-mass spectroscopy
methanol
nuclear magnetic resonance spectroscopy
phosphorus oxychloride
trifluoroacetic acid
THF
tetrahydrofuran
TLC
thin layer chromatography
UP-LCMS ultra-performance liquid chromatography-mass spectrometry
1. Introduction
1.1 Small molecule fluorophores
Fluorescent tools are indispensable in the imaging, study, and under-
standing of biological systems. Compared to fluorescent proteins, synthetic
fluorophores are often characterized by their smaller size, greater brightness,
and higher photostability, allowing for the elucidation of biological informa-
tion without the need for genetic engineering (Terai & Nagano, 2008).
These attributes make synthetic small molecule fluorophores essential tools
that can be employed as cellular stains, biomolecular labels, environmental
indicators, and biosensors. However, development of new fluorescent dyes
and probes presents a challenge due to the often-unpredictable relationship

Quinoline-based fluorescent small molecules
311
between molecular structure and photophysical properties. Furthermore,
there is the challenge of developing facile syntheses of core scaffolds that
are amenable to late-stage functionalization and easy derivatization
(Finney, 2006). As a result, most fluorophore development has focused
on a limited number of core scaffolds such as BODIPY, coumarin, fluores-
cein, rhodamine, and cyanine (Lavis & Raines, 2008). The perfect fluo-
rophore has yet to be developed, as many existing probes suffer from cell
toxicity, impermeability, low aqueous solubility, and poor sensitivity.
Therefore, a demand exists for simple scaffolds that can allow researchers
to combine rational design and a high-throughput approach (Vendrell,
Zhai, Er, & Chang, 2012).
1.2 Environmentally sensitive dyes
Many biologists aim to study processes in which relatively small changes
to the environment, such as fluctuations in pH, interactions between
biomolecules, or the presence of ions and other analytes, can have major
downstream effects. Therefore, dyes that are sensitive to changes in the
microenvironment are particularly useful. Dyes that can respond to local
changes through variations in either fluorescence or intensity are called
chromogenic or fluorogenic, respectively.
One subcategory of chromogenic or fluorogenic dyes undergoes a change
in fluorescence as the dye’s microenvironment’s polarity or viscosity changes.
This phenomenon is known as solvatochromism. Solvatochromism can
be caused by a number of factors, including intramolecular charge transfer,
intramolecular proton transfer, conformational changes, aggregation, or
combinations thereof. Solvatochromic dyes have been applied to the study
of lipid rafts, apoptosis, and interactions with proteins or nucleic acids
(Klymchenko, 2017).
A second subcategory is pH sensors, which can be excited or emit at
different wavelengths depending on the presence or absence of a proton
(Han & Burgess, 2010; Shi, Li, & Ma, 2014). Changes in cellular pH are
associated with a number of normal and abnormal processes, including
ion transport, phagocytosis, inappropriate cell growth, function, or division,
and endocytosis. Furthermore, pH is not necessarily consistent across the
cell, with some compartments, such as the lysosome, being more acidic than
others. pH sensing dyes can be used to develop ratiometric spectroscopic

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methods, in which the dye can be observed under two wavelengths and the
ratio of the signal can be calibrated and used to determine the exact pH across
different cellular compartments.
With this in mind, quinoline-based dyes, which contain a nitrogen
heteroatom (a site for analyte recognition) and can be functionalized
with push-pull donor/acceptor moieties (for intracellular charge transfer),
have immense potential as biological sensors (Fahrni & O’Halloran, 1999;
Hanaoka, Kikuchi, Kojima, Urano, & Nagano, 2004; Klymchenko, 2017;
Li, Zhu, Xue, & Jiang, 2013; Meeusen, Tomasiewicz, Nowakowski, &
Petering, 2011; Nowakowski & Petering, 2011). Though quinine, the
first fluorescent molecule to be studied, was reported in 1845, quinoline-
based fluorophores are understudied due to a myriad of synthetic challenges
(Herschel, 1845; Kaufman & Ru´veda, 2005; Lavis & Raines, 2008).
Previously reported synthetic routes require harsh conditions, complex or
inefficient purification, and specialty glassware. Furthermore, they result in
moderate yields that may be difficult to reproduce (Eisenhart, Howland, &
Dempsey, 2016; Nasr, Drach, Smith, Shipman, & Burckhalter, 1988).
We report a facile, high-yielding, one-pot synthesis of a key quinoline
precursor that can be performed on a gram-scale (Scheme 1). This critical
scaffold contains an electron-donating motif that acts as a polarization
domain and two tunable domains for late-stage chemical modification
via either a regioselective or sequential Suzuki-Miyaura coupling ( Jun,
Petersson, & Chenoweth, 2018). We refer to the resulting scaffold as
dimethylamino quinoline (DMAQ). We also present optimized, selective
syntheses for both mono- and bisarylated quinolines to create a library
of dyes with varied photophysical properties (Schemes 2 and 3). This
method also describes a procedure for high-throughput analysis of this
library and for performing live cell imaging of a hit compound in HeLa
cells (Fig. 1).
Scheme 1 Synthesis of core scaffold 3, containing a polarization domain (highlighted in
blue) and two tunable domains (highlighted in orange).

Quinoline-based fluorescent small molecules
313
2. Chemical synthesis
2.1 Reagents, equipment, and general procedures
Chemical reagents are listed below. Solvents were used directly from a
JC Meyer Solvent System (Laguna Beach, CA), and Mili-Q filtered
(18MΩ) water was used for all solutions unless otherwise stated
(Millipore; Billerica, MA, USA). Liquid chromatography-mass spectro-
metry (LCMS) was performed using a Waters Acquity Ultra Performance
LC connected to a single quadrupole detector (SQD) mass spectrometer
(Milford, MA, USA). High resolution mass spectrometry (HRMS) was
obtained with a Waters LCT Premier XE, time-of-flight LCMS with
electrospray ionization (ESI). Nuclear magnetic resonance (NMR) spectra
were acquired using Bruker UNI-500 or AVII-500B and processed with
either MestReNova or TopSpin software. Analytical HPLC was performed
using a JASCO-FC-2088-30 HPLC fitted with a Phenomenex Luna
Omega 5μm PS C18(2) 100A; 250ꢀ4.60mm column.
2.1.1 Equipment and instrumentation
Fume Hood
Rotary evaporator system
Stirring Hot Plate (IKA RCT basic; No: 003810001)
Glovebox
Pipettes (100, 1000μL)
Pipette tips
Nitrogen or argon gas (Airgas NI R200SS or AR 300)
NMR, HRMS, and analytical HPLC for analysis
Column chromatography for purification
Automated Flash chromatography (Teledyne ISCO Combiflash Rf+)
Silica gel (Silicycle silica gel 55–65A° pore diameter)
Sodium sulfate (Na2SO4)
Scintillation vials (4, 10, and 40mL)
Green Open Top SURE-LinkTM Polypropylene Cap (Chemglass Life
Sciences CG-4909-05)
Stirbars
Teflon tape (Fisherbrand™ Pure Low Density PTFE Thread Seal Tape,
15-078-260)
TLC silica gel plates (60G F254, Millipore Sigma)
TLC chamber

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Handheld UV lamp with longwave (365nm) and shortwave (254nm)
Potassium permanganate (KMnO4) staining solution
1.5g KMnO4 +10g K2CO3 +1.25mL 10% NaOH+200mL water
Syringe
Needle
Balloon
pH testing strips (Hydrion (93) S/R Inst-Chek Disp 0.0-13.0)
Separatory funnel
2.1.2 Chemical reagents
N,N-dimethyl-1,3-phenylenediamine dihydrochloride (CAS: 536-46-9;
Acros Organics AC204350250)
Potassium carbonate (CAS: 584-08-7; ACROS 424081000)
Diethyl malonate (CAS: 105-53-3; Alfa Aesar A1546836)
Phosphorus oxychloride [Highly Corrosive] (POCl3) (CAS 10025-87-3;
Acros Organics 105-53-3)
Brine (saturated aqueous sodium chloride)
Sodium sulfate (Na2SO4) (CAS 7757-82-6; Aldrich 822286)
Tetrahydrofuran, anhydrous, degassed (from the solvent system)
Dioxane, anhydrous, degassed (from the solvent system)
Degassed water
Ethyl acetate (EtOAc)
Hexanes, reagent grade
Diphenyl ether (CAS: 101-84-8; Aldrich 240834)
Dichlorobis(triphenylphosphine)palladium(II) dichloride (PdCl2(PPh3)2)
(CAS: 13965-03-2; Aldrich 208671)
(Hetero)aryl boronic acids
2.2 Synthesis of 2,4-dichloroquinoline core
The DMAQ scaffold can be readily synthesized through a one-pot conden-
sation of 3-(dimethylamino) aniline and diethyl malonate to form 2
(Scheme 1). Though previously published syntheses involve running the
cyclo-condensation reaction neat, this results in a heterogeneous reaction,
which leads to poor mixing and low yields. We address this challenge by
performing the reaction in diphenyl ether, a nonpolar, high boiling
(258°C) solvent that is found to be inert to both the cyclo-condensation
and chlorination reaction. Compound 3 is then prepared by addition of
POCl3 prior to purification.

Quinoline-based fluorescent small molecules
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(a) N,N-dimethyl-1,3-phenylenediamine dihydrochloride (5.70g,
270mmol, 1.0 equiv.) is placed in a dry 500mL Erlenmeyer flask
and dissolved in 100mL of deionized water.
(b) Neat K2CO3 (ꢁ15g) is added until pH approaches 10–11.
(c) The substance is then extracted with EtOAc (3ꢀ100mL) in a 500mL
separatory funnel.
(d) The organic layers are then combined and washed once with
200mL brine.
(e) After drying over Na2SO4 for 15min, the liquid is concentrated using
a rotary evaporator, giving the 1 as a brown oil (3.63g). (See Synthetic
note 1.)
(f ) In
a
40mL scintillation vial, free-based N,N-dimethyl-1,
3-phenylenediamine (1.20g, 8.81mmol, 1.0 equiv.) is dissolved in
2.0mL diphenyl ether (See Synthetic note 2).
(g) Diethyl malonate (1.55g, 9.69mmol, 1.1 equiv.) is added, the threads
of the cap are wrapped tightly with Teflon tape for a tight seal, and
the reaction is capped with a Green Open Top SURE-Link™
Polypropylene Cap.
(h) The reaction vial is then heated and stirred at 200°C for 4h
(See Synthetic note 3) or until TLC shows complete formation of 2
(Rf ¼0.34 in 10% MeOH in DCM or 0.0 in 1:1 EtOAc/hexanes).
(i) The reaction vial is cooled to room temperature.
(j) Without isolation or purification of 2, a syringe is used to add POCl3
(13.5g, 88.1mmol, 10.0 equiv.) to the reaction vial.
(k) The reaction mixture is stirred at 100°C for 1–3h. Upon conversion
of starting material to product (Rf ¼0.47, 1:5 EtOAc/hexanes) the
vial is removed from heat and cooled over ice for 30min.
(l) Following cooling, ice water is added slowly without removing the
vial from the ice bath (See Synthetic note 4). As the ice melts, the solu-
tion will begin to bubble and smoke violently.
(m) After the exotherm settles and the vial is cool enough to handle, the
reaction mixture is carefully poured into a 500mL flask containing
additional ice and slowly neutralized with K2CO3.
(n) The crude mixture is then extracted with EtOAc (3ꢀ100mL).
(o) The organic layers are combined and washed once with brine, then
dried over sodium sulfate for 15min and concentrated in vacuo.
(p) The product is purified by flash column chromatography (0%–30%
ethyl acetate in hexanes). Diphenyl ether elutes at 100% hexanes,
and 3 is obtained as a yellow, needle-like, crystalline solid (2.03g,
95.6%).

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2.3 Synthesis of monoarylated quinolines, 4a-n
A regioselective Suzuki coupling was optimized for selectivity of addition
at the 2-position over the 4-position of 3. The optimized conditions were
extended to couple 14 boronic acids of varying electronic and steric prop-
erties. This reaction tolerates phenyl boronic acids containing a range of
electron-donating, -withdrawing, and -neutral functional groups, such as
methoxy, dimethylamino, fluoro, cyano, and a Boc-protected primary
amine (Scheme 2). Further detail about this reaction’s optimization and
Scheme 2 Substrate scope for monoarylated DMAQ, 4.

Quinoline-based fluorescent small molecules
317
substrate scope, as well as characterization and isolation information for
4a-n, can be found in the supplementary section of Jun et al. (2018).
(a) Under inert atmosphere, such as inside of a glovebox (see Synthetic
note 5), 696mg 3 (2.88mmol) is dissolved in 1.6mL of dry, degassed
THF to make a stock solution.
(b) 100μL of stock solution is then added to each 4.0mL scintillation vial
(43.5mg, 0.18mmol, 1.0 equiv.) that has been fitted with a small stir bar
and wrapped with Teflon tape.
(c) To the stock solution is added PdCl2(PPh3)2 (6.3mg, 0.009mmol,
0.05 equiv.), boronic acid (0.198mmol, 1.1 equiv.), and
1.4mL THF.
(d) K2CO3 is then dissolved in 361μL degassed water and added to the
THF solution, giving a final concentration of 0.1M.
(e) The reaction mixture is sealed under nitrogen and removed from the
glovebox to stir at 65°C.
(f ) As the reaction proceeds, the vial can be cooled and returned to the
glovebox. An aliquot is removed for monitoring by TLC and LCMS
(avoiding the introduction of oxygen during the course of the reaction)
and the reaction vessel is again removed from the glovebox to be heated
and stirred.
(g) The reaction will proceed to completion in a period of 5–48h,
dependent on the boronic acid being coupled (see Synthetic note 6).
Upon reaction completion, the reaction vial is cooled and transferred
to a 100mL separatory funnel, to which 30mL each EtOAc and
deionized water are added.
(h) The organic layer is extracted with EtOAc and rinsed once with brine
solution.
(i) The organic layer is then dried over Na2SO4 for 15min before being
concentrated in vacuo and purified by column chromatography.
2.4 Synthesis of bisarylated quinolines, 5a-i
Further detail about this reaction’s substrate scope, as well as characterization
and isolation information for 5a-i, can be found in the supplementary
section of Jun et al. (2018).
(a) Under inert atmosphere, such as inside of a glovebox under nitrogen,
200mg 3 (0.83mmol) is dissolved in 1.0mL dry, degassed dioxane to
create a stock solution.
(b) 100μL of stock solution is then added to each 4.0mL scintillation vial
(20mg, 0.083mmol, 1.0 equiv.) that has been fitted with a small stir bar
and sealed with Teflon tape.

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Rachel M. Lackner et al.
Scheme 3 Synthesis of bisarylated DMAQ via a one-pot, two step Suzuki cross-coupling.

Quinoline-based fluorescent small molecules
319
(c) To the stock solution is added PdCl2(PPh3)2 (2.9mg, 0.004mmol, 0.05
equiv.), first boronic acid (0.087mmol, 1.05 equiv.), and 664μL of
dioxane.
(d) K2CO3 is then dissolved in 165.9μL degassed water and added to the
dioxane solution, giving a final concentration of 0.1M.
(e) The reaction vial is sealed under nitrogen and removed from the
glovebox to stir at 65°C for 24h.
(f ) After the first coupling has reached completion, the solution is cooled
to room temperature before being returned to an inert atmosphere for
the addition of a second boronic acid (0.249mmol, 3.0 equiv.). The vial
must be resealed with fresh Teflon tape.
(g) The temperature is then increased to 85°C and the vial is stirred under
inert atmosphere for an additional 24h, with monitoring by TLC
and LCMS.
(h) Upon completion of the second coupling, the reaction vial is cooled
to room temperature and the contents are transferred to a 100mL
separatory funnel.
(i) 30mL EtOAc and water are added, and the organic layer is extracted
before being dried over Na2SO4.
(j) Solvent is then removed in vacuo and the compound is purified by
column chromatography (Scheme 3).
2.5 Synthetic notes
(1) 1 may react with acetone, so it is important to ensure that all glassware
is dry prior to free-basing and to avoid using acetone to transfer
compound.
(2) Due to diphenyl ether’s high melting point (ꢁ27 °C), care must be
taken when dispensing liquid diphenyl ether from a syringe, as it
may solidify at room temperature, causing a buildup of pressure that
can cause the needle to separate from the syringe.
(3) We found that we could push the equilibrium of the cyclo-
condensation reaction forward by removing EtOH without the use
of special glassware. To do this, the plunger of a 2 mL syringe is
removed, and a balloon is stretched across the top and sealed with para-
film and a rubber band. The syringe is fitted with a long needle and the
needle is pushed through the cap, making sure it does not touch

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the reaction solution. Liquid EtOH will condense in the syringe as the
reaction proceeds.
(4) Care must be taken to slowly add ice water to the reaction vessel
and to ensure that the vessel is no more than half-full to minimize
the danger of exothermic quenching of corrosive POCl3. We find that
adding a minimal amount of ice and allowing several minutes before
adding more is necessary, as quenching can take several minutes to
begin. Adding excessive water or ice too quickly can cause the vial
to bubble over or splash.
(5) If not using a glove box, the catalyst is bench stable and can be handled
in air prior to dissolving in solution, at which point care must be taken
to ensure maintenance of an air-free reaction vessel.
(6) For more detailed information regarding the reaction times and yields
for each boronic acid, see Jun et al. (2018).
3. Plate reader analysis of DMAQ derivatives
Having developed a straightforward synthesis of a library of DMAQ
derivatives, we desired a method by which to quantitatively evaluate the
probes in a high-throughput manner. The photophysical properties of these
probes were anticipated to be highly impacted by their environment, due
both to the quinoline nitrogen as a site for analyte recognition and to the
push-pull scaffold. The protocol below describes a multi-well assay we
employed to screen our library of compounds and assess their response to
changing solvent polarity and pH in a high-throughput manner. We chose
to excite our compounds at 405nm, as this is the wavelength of a commonly
used laser line in confocal microscopy. However, the wavelength of excita-
tion can be optimized based on the compound of interest. This method
can be extended to monitoring the fluorescent response to a variety of
other elements, such as metals, peptides, DNA, and other biological macro-
molecules prior to application in biological systems.
3.1 Reagents and equipment
Plate reader:
Tecan M1000 plate reader (Mannedorf, Switzerland)
Greiner 96 Flat Bottom Black Polypropylene plate
Greiner 96 Flat Bottom Black Polystyrene plate
Thermo Scientific™ Nunc™ Sealing Tapes (Thermo Scientific™
232698)

Quinoline-based fluorescent small molecules
321
Solvents:
DMSO
Water
Toluene
DMF
DMSO
DCM
EtOH (200 proof )
Trifluoroacetic acid
Aluminum oxide, activated, basic, Brockmann I
Buffers:
Hydrochloric acid (10mM, pH 2)
Citric acid/phosphate (10mM, pH 4)
Citric acid/phosphate (10mM, pH 6.9)
Phosphate (10mM, pH 7.3)
Carbonate (10mM, pH 10)
Sodium hydroxide (10mM, pH 11)
Miscellaneous
Pipettes (10, 100, 1000μL)
Pipette tips
Eppendorf tubes
3.2 Procedure
(a) Solvents are prepared. For each plate, a minimum of 2.0mL of each
solvent is required, but it is advisable to prepare an excess of stock to
accommodate evaporation or loss during transfer. For neutral and acidic
DCM: 6–10mL of DCM are dried over activated alumina for several
minutes, to neutralize. To 3.0mL of DCM is added 3μL TFA (0.1% by
volume) to acidify.
(b) 1–2mg of each dye are dissolved in DMSO for a final concentration
of 5mM. The amount of dye stock required will depend on the
number of conditions being screened. For each assay of 12 conditions,
a minimum of 30μL of each dye will be required, but it is advi-
sable to prepare an excess of stock to accommodate loss during
transfer.
(c) Using more than one plate if necessary, each row is dosed with 2.5μL
of dye stock solution, ensuring the pipette tip is changed each time a
new dye is used. (If dye is added before solvent, the same tip can be
used across a row.) If using organic solvents or high temperatures, a

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polypropylene plate must be used. If using aqueous solutions and
buffers and moderate temperatures, polystyrene is sufficient.
(d) Each well is then dosed with 250μL solvent and mixed well, changing
pipette tips each time. Particularly volatile solvents (such as DCM) are
added last to avoid evaporation.
(e) At this point, the plate can be visualized under a hand-held UV lamp.
The plate is then covered with sealing tape prior to analysis to avoid
evaporation and contamination. The sealing tape will be removed
for plate reader analysis, though it should be noted that evaporation
prior to and during analysis can impact the results. Therefore, intensity
values should only be treated as approximate.
(f ) Regardless of absorption maxima of the individual dyes, each com-
pound is excited at 405nm. Emission is scanned from 420 to 780nm
at a constant temperature of 20°C. Details of additional settings can
be found in the supplemental information of Jun et al. (2018).
4. Cell imaging of an environmentally sensitive dye
Following in vitro library analysis, 5a was found to exhibit interesting
pH-sensing properties, with a bathochromic shift in emission of 168nm
Fig. 1 Fluorescence spectrum and live cell imaging of 5a. (A) Normalized UV absorption
(dotted) and emission (solid) of 5a at pH 7. (B) Normalized UV absorption and emission
of 5a at pH 4. (C, F) Merged image of magenta (405ex/FITCem) and cyan (488ex/Cy5em)
channels following incubation with 5a. Scale bar represents 10μm. (White box in
(C) represents area shown in greater detail in F–H.) (D, G) Grayscale image of 405ex/
FITCem (525/36nm)a channel. (E, H) Grayscale image of 488ex/Cy5em (700/72nm)a
a
channel. Filter center wavelength/full width at half maximum.

Quinoline-based fluorescent small molecules
323
from pH 7 to 4 (Fig. 1A-B). We therefore turned our attention to demon-
strating this compound’s dual emissive properties in cellulo (Fig. 1C–G).
4.1 Reagents and equipment
HeLa cells
Biosafety cabinet
Incubator with CO2
Pipettes (10, 100, 1000μL)
Pipette tips
Eppendorf tubes
Glass bottom dish, 35mm, No. 1.5 Coverslip, Poly-D-Lysine Coated
(MatTek, P35GC-1.5-14-C)
DMSO, anhydrous, 99.8+% (CAS: 68-68-5; Afla Aesar 43998)
Growth media
Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, 11965084)
10% Fetal bovine serum (FBS) (Life Technologies, 16000-044)
1% Pen/Strep (Invitrogen, 15140122)
Dulbecco’s phosphate-buffered saline (DPBS) (Invitrogen 14040117)
Microscope compatible with live-cell imaging at 405 and 488nm (Nikon
Eclipse Ti2 with Yokogawa CSU-X1 spinning disk confocal).
Imaging media
DMEM, no phenol red (Invitrogen, 21063029) OR Leibovitz-15
(L15) CO2-independent media, no phenol red (Life Technologies
11415064)
10% Fetal bovine serum (FBS) (Life Technologies, 16000-044)
1% Pen/Strep (Invitrogen, 15140122)
5mM dye stock in DMSO
Trypsin 0.25% EDTA (Invitrogen, 25200056)
4.2 Procedure
(a) 1–2mg 5a is dissolved in DMSO to give a concentration of 5mM and
aliquots are divided among several Eppendorf tubes. The dye can be
stored at 4°C and warmed to 37°C when ready for use. Avoid exces-
sive freeze/thaw cycles.
(b) HeLa cells are cultured in glass-bottomed MatTek dishes coated with
poly-D-Lysine until they reach 60%–80% confluency.

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(c) In a 5mL Falcon tube, 2μL of 5mM 5a in DMSO is added to 2mL
growth media and mixed well to give a final concentration of 5μM.
(d) Growth media is removed from cells and exchanged for media
containing 5a.
(e) Cells are incubated under CO2 for 3h.
(f ) Cells are washed with warm growth media three times.
(g) Growth media is replaced with warm imaging media (DMEM, no phe-
nol red if using an imaging chamber connected to CO2 or Leibovitz-15
if not).
(h) The dish is mounted onto the microscope and a population of multiple
cells is located, avoiding overly crowded populations. CO2 and incu-
bation can be turned on to allow for long-term imaging.
(i) The dye is excited at both 405nm and 488nm (10%–20% laser power)
and emission is collected at a range of wavelengths. We found that
405ex/FITCem and 488/Cy5em showed optimal dual emission
(Fig. 1) but this will be dye and microscope dependent. Care should
be taken not to photobleach the dye.
4.3 Analysis
Images can be analyzed using NIS-Elements (Nikon’s proprietary image
analysis software) or using the open-source software FIJI, provided by the
NIH at https://imagej.net/Fiji.
5. Summary and conclusions
In this chapter, we have presented detailed procedures for synthesiz-
ing, analyzing, and employing a library of DMAQ derivatives for live cell
imaging. Ease of synthesis can be a barrier to biological utility, and we
therefore offer a simple, straightforward synthesis of a medically relevant
quinoline core with excellent tunability and varied photophysical proper-
ties. This core can be modified at the 2-position for structural diversity and
the 4-position for further late-stage functionalization, allowing for the
facile generation of a library of mono- and bisarylated dyes with emission
spectra that collectively span the entire visible light region. These com-
pounds exhibit intriguing pH-sensing and solvatochromic qualities and
emphasize the utility of a high-throughput approach for library generation
and analysis. We have therefore begun to probe an underexplored scaffold

Quinoline-based fluorescent small molecules
325
Fig. 2 Workflow for combining rational design and high-throughput approach for fluo-
rophore development.
that has great potential as a biologically relevant dye. Furthermore, we
present a strategy for developing and analyzing a library of compounds
from underexplored dye families in a high-throughput manner (Fig. 2).
Our method, which combines rational design with a combinatorial
approach, can easily be extended to other dye scaffolds.
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