Dibenzothiophene Sulfone Derivatives as Plasma Membrane Dyes

Article abstract of DOI:10.1111/php.13175

Dibenzothiophene 5,5-dioxide (DBTOO) derivatives have recently been shown to processes utility as fluorescent cell dyes. In an effort to extend the functionality of DBTOO-based dyes to include the visualization of cellular membranes, two lipophilic DBTOO were synthesized and their ability to incorporate into the plasma membrane of HeLa cells was examined by fluorescent microscopy. The photophysical properties of the two new DBTOO derivatives were determined and both have good fluorescent quantum yields and a visible blue emission. Due to agreeable wavelengths of excitation and emission, a standard 4′,6-diamindino-2-phenylindole (DAPI) filter set worked well with these dyes. After co-staining, it was confirmed that both DBTOO dyes localized in the plasma membrane. The quality of the overlap was quantified using Pearson correlation coefficient, which indicated a strong overlap between the DBTOO dyes and the standard plasma membrane dye. The novel dyes also displayed relatively low toxicity to the HeLa cells with IC₅₀ between 10 and 100?μm. Thus, this work reports a new use of DBTOO derivatives as fluorescent microscopy stains.

Full text of DOI:10.1111/php.13175

DR. RYAN MCCULLA (Orcid ID : 0000-0003-3381-8880)
Article type : Research Article
Dibenzothiophene Sulfone Derivatives as Plasma Membrane Dyes
John T. Petroff II, Scott Grady, Christopher K. Arnatt, and Ryan D. McCulla*
3501 Laclede Ave., Department of Chemistry, Saint Louis University, St. Louis, MO 63103,
United States of America.
*Corresponding author e-mail: ryan.mcculla@slu.edu (Ryan McCulla)
This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as doi: 10.1111/PHP.13175
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ABSTRACT
Dibenzothiophene 5,5-dioxide (DBTOO) derivatives have recently been shown to processes
utility as fluorescent cell dyes. In an effort to extend the functionality of DBTOO based dyes to
include the visualization of cellular membranes, two lipophilic DBTOO were synthesized and their
ability to incorporate into the plasma membrane of HeLa cells was examined by fluorescent
microscopy. The photophysical properties of the two new DBTOO derivatives weredeterminedand
both have good fluorescent quantum yieldsand a visible blue emission. Due to agreeable wavelengths
of excitation and emission, a standard 4’,6’-diamindino-2-phenylindole (DAPI) filter set worked well
with these dyes. After co-staining,itwas confirmed that bothDBTOO dyes localized in the plasma
membrane. The quality of the overlap was quantified using Pearson correlation coefficient, which
indicated a strong overlap between the DBTOO dyes and the standard plasma membrane dye. The
novel dyes also displayed relatively low toxicity to the HeLa cells with IC50 between 10 µM and
100µM.Thus, this work reports a new use of DBTOO derivatives as fluorescent microscopy stains.
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INTRODUCTION
A large variety of options exist for staining tissue and cells, which allows for many possible
solutions to circumvent the limitations of any one method (1). These methods involve antibodies,
fluorescent proteins, or materials that allow fluorescent imaging with their own set of advantages or
disadvantages (2, 3). For small molecule dyes, the innate limitations of the dye’s photophysics are
often the cause that disallow use of the dye. This problem is described by Terai and Nagano, who note
the difficulty of predicting a given molecule’s photophysics, which motivates the need to synthesize
new small molecule fluorescent molecules as candidate fluorescent dyes (4). In response to this
prompt, dibenzothiophene 5,5-dioxides (DBTOOs) based microscopy dyes were first reported in 2018
(5). Figure 1 shows the DBTOO fluorophore. The report described dyes consisting of the DBTOO
backbone with unsymmetrical substitutions of a polar sulfonate and nonpolar aryl group. These dyes
localized in the nucleus of HeLa cells. These dyes showed low toxicity and worked well with a
standard 4’,6’-diamindino-2-phenylindole (DAPI) filter set.
>Figure 1<
In addition to fluorophore-lipid conjugates and lipid-binding probes, lipophilic dyes are
commonly used for the visualization of lipid membranes (6). Traditional lipid soluble diazo dyes,
such as Oil Red or Sudan Black, are sensitive to preparation conditions and can only be used with
fixed samples (7). Other dyes that localize within plasma membranes include Nile Red and various
BODIPY derivatives (8, 9). The natural hydrophobicity of these dyes promotes the uptake of these
fluorophores into lipid membranes. To improve the selectivity of these dyes, the modification of
BODIPY to a fatty acid has been used (10). DBTOO dyes have a different emission and spectral
profiles than existing lipid soluble dyes.
To further extend the utility of DBTOO fluorophores in microscopy, new DBTOO based dyes
were needed. In this work, the DBTOO backbone was functionalized with a butanoic acid or a
combination of a butanoic acid and an n-octyl group to create two new dyes. The hypothesis was the
addition of these groups to DBTOO fluorophore would lead to localization of the new DBTOO dyes
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within lipid plasma membranes. The two new DBTOO derivatives prepared for this work were
chosen as analogs to other fluorophores that have been verified to function as lipophilic dyes (1).
MATERIALS AND METHODS
Materials. All chemicals were purchased from Alfa Aesar, Oakwood Chemical, Ark Pharm,
ThermoFisher Scientific, and Sigma-Aldrich. They were used as received without further purification.
Flash chromatography was performed using a Biotage Horizon with silica gel (SiliaFlash P60)
1
13
purchased from SiliCycle. H and C NMR spectra were gathered on a Bruker DRX-400 in either
DMSO-d6 or CDCl3. High-resolution mass spectra were measured using a ThermoScientific Q
Exactive Orbitrap equipped with a Nano ESI ionization source. Absorption spectra were recorded
using a Shimazdu UV-1800UV–Vis spectrophotometer using samples contained in 10 x 10 mm
quartz cell at a concentration of 0.01 mM. Fluorescence spectra were gathered on a Shuimazdu RF-
5301 PC in a 10 x 10 mm quartz cuvette. Fluorescent microscopy images were taken on a Leica DM
400 B microscope with a DFC3000G camera using a DAPI filter set. Images were processed using
ImageJ.
4-(Dibenzo[b,d]thiophen-2-yl)-4-oxobutanoic acid (2). Dibenzothiophene (1) (1.008 g, 5.5 mmol)
was combined with succinic anhydride (602 mg, 5.9 mmol) in a sealed two neck round bottom flask
under nitrogen atmosphere. To the sealed flask, 7 mL of 2:1 dichloroethane(DCE)/nitrobenzene was
injected by syringe. The mixture was stirred until a homogeneous solution was formed. The flask was
then cooled to 0 °C by an ice water bath. Upon cooling, the AlCl3 (1.494 g, 11.2 mmol) was added
slowly to the stirring solution over several minutes. Once the AlCl3 addition was complete, the
solution was allowed to stir at room temperature for five hours. After the fifth hour of stirring at room
temperature, the solution was warmed to 60°C for 30 minutes. The solution was then allowed to cool
to room temperature. It was then poured over HCl acidified water and washed with dichloromethane
(DCM) 3 x 75 mL. The combined organic layers were dried with anhydrous magnesium sulfate
(MgSO4) then the solution was evaporated under reduced pressure affording a dark brown oil. The oil
was dissolved in minimal refluxing 2:1 ethyl acetate (EtOAc)/chloroform (CHCl3) then placed in a
freezer overnight. The resulting solid was recrystallized twice more using the same 2:1 EtOAc/CHCl3
1
method before affording 733 mg of a slightly orange solid (47% yield) (11). H NMR (400 MHz,
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Chloroform-d) δ = 8.80 (d, J = 1.3 Hz, 1 H), 8.29 - 8.23 (m, 1 H), 8.09 (dd, J = 1.7, 8.4 Hz, 1 H), 7.94
(d, J = 8.4 Hz, 1 H), 7.91 - 7.86 (m, 1 H), 7.57 - 7.49 (m, 2 H), 3.52 - 3.42 (m, 2 H), 2.94 - 2.86 (m, 2
H)13C NMR (Chloroform-d, 101MHz): δ (ppm) 197.4, 177.1, 144.9, 139.7, 135.7, 135.2, 133.0,
127.5, 125.9, 125.0, 123.0, 122.9, 121.9, 121.5, 33.4, 27.9.
4-(Dibenzo[b,d]thiophen-2-yl)butanoic acid (3). Zinc powder (2.15 g), mercuric chloride (HgCl2)
(225 mg), HCl (100 μL), and deionized (DI) water (2.75 mL) were combined in a 50 mL round
bottom flask and stirred for five minutes. Due to HgCl2 being highly toxic, extreme care and
ventilation was used. After the stirring, the liquid was decanted. To the flask with the remaining zinc
amalgam, DI water (1.4 mL), HCl (325 μL), toluene (1.8 mL) and 2 (1.007 g, 3.5 mmol) were added.
The solution was refluxed for 40 hours. During the reflux, approximately 300μL of HCl was added
approximately every 7 hours for a total of 5 additions. After reflux, the solution is poured over 30 mL
of water acidified by 1.5 mL of HCl. The solution was washed with 3 x 50 mL DCM. The combined
organic washes were dried with anhydrous MgSO4 and evaporated under reduced pressure with
Celite. The resulting homogenous solid mixture was purified by normal phase flash chromatography
using 1:1 EtOAc:hexanes with 0.1% acetic acid. The collected fractions were evaporated under
reduced pressure to yield an off-white oil (567.2 mg, 58% yield) which eventually solidified (11). 1H
NMR (400 MHz, Chloroform-d) δ = 8.18 - 8.12 (m, 1 H), 8.01 - 7.94 (m, 1 H), 7.89 - 7.81 (m, 1 H),
7.78 (d, J = 8.2 Hz, 1 H), 7.50 - 7.41 (m, 2 H), 7.31 (dd, J = 1.6, 8.2 Hz, 1 H), 2.88 (t, J = 7.6 Hz, 2
H), 2.49 - 2.39 (m, 2 H), 2.09 (quin, J = 7.4 Hz, 2 H)13C NMR (Chloroform-d, 101 MHz): δ (ppm)
177.1, 139.8, 137.6, 137.2, 135.8, 135.4, 127.6, 126.6, 124.3, 122.9, 122.7, 121.5, 121.3, 35.0, 32.8,
26.6.
4-(5,5-Dioxidodibenzo[b,d]thiophen-2-yl)butanoic acid (4). 4-(Dibenzo[b,d]thiophen-2-yl)butanoic
acid (3) (86 mg, 0.3 mmol) was combined with 10 mL of DCM. To this solution, meta-
Chloroperoxybenzoic acid (mCPBA) (77%) (279 mg, 1.6 mmol) was added. The solution was stirred
overnight. After stirring, the solution was poured over 50 mL of saturated sodium bicarbonate water.
An additional 20 mL of DCM was added. The organic layer was washed with 4 x 30 mL saturated
sodium bicarbonate water. The organic layer was separated, dried with anhydrous MgSO4, and
filtered. The dry organic layer was evaporated under reduced pressure with Celite. The resulting
homogenous solid mixture was purified by normal phase flash chromatography using 1:1
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EtOAc:hexanes with 0.1% acetic acid. The collected fractions were evaporated under reduced
1
pressure to yield a white solid (63.8 mg, 66%). H NMR (400 MHz, DMSO-d6) δ = 12.10 (br. s., 1
H), 8.20 (d, J = 7.7 Hz, 1 H), 8.06 (s, 1 H), 7.95 (d, J = 7.5 Hz, 1 H), 7.88 (d, J = 7.9 Hz, 1 H), 7.80 (t,
J = 7.5 Hz, 1 H), 7.65 (t, J = 7.6 Hz, 1 H), 7.48 (d, J = 8.1 Hz, 1 H), 2.76 (t, J = 7.8 Hz, 2 H), 2.27 (t, J
= 7.3 Hz, 2 H), 1.90 (quin, J = 7.6 Hz, 2 H)13C NMR (DMSO-d6, 101 MHz): δ(ppm) 174.1, 149.2,
137.4, 134.7, 134.4, 131.2, 130.9, 130.9, 130.9, 122.6, 122.5, 121.9, 34.5, 33.0, 25.8 HRMS (ESI)
calculated for C16H13O4S- m/z: 301.0534, found m/z: 301.0540.
2-Octyldibenzo[b,d]thiophene
(6).
To
a
three
neck
round
bottom
flask,
2-
bromodibenzo[b,d]thiophene (5) (5.04 g, 19.2 mmol) and 1,3-Bis(diphenylphosphino)propane
nickel(II) chloride [NiCl2(dppp)] (517.0 mg, 0.9 mmol) were added. The flask was sealed and purged
with nitrogen gas for 30 minutes. To the flask, and while remaining under a nitrogen atmosphere, 500
mL of dry tetrahydrofuran (THF) was added. The flask was then cooled to 0°C with an ice water bath.
Once cooled 12.3 mL of octylmagnesium bromide (2M in ether) was slowly added by syringe. The
solution was then stirred overnight at room temperature. Following the stirring, the solution was
evaporated under reduced pressure with Celite. The resulting homogenous solid mixture was purified
by flash chromatography using hexanes. The fractions associated with the product were evaporated
under vacuum to afford a colorless oil (3.65 g, 64%). 1H NMR (DMSO-d6, 400MHz): δ (ppm) 8.31 -
8.37 (m, 1 H), 8.18 (s, 1 H), 7.96 - 8.03 (m, 1 H), 7.90 (d, J = 8.2 Hz, 1 H), 7.45 - 7.54 (m, 2 H), 7.36
(d, J = 8.1 Hz, 1 H), 2.76 (t, J = 7.6 Hz, 2 H), 1.67 (t, J = 7.2 Hz, 2 H), 1.15 - 1.39 (m, 10 H), 0.79 -
0.89 (m, 3 H)13C NMR (DMSO-d6, 101 MHz): δ (ppm) 139.2, 138.8, 135.8, 135.1, 135.0, 127.8,
126.9, 124.6, 123.0, 122.7, 121.9, 121.4, 35.1, 31.3, 31.2, 28.8, 28.7, 28.6, 22.1, 13.9.
4-(8-Octyldibenzo[b,d]thiophen-2-yl)-4-oxobutanoic acid (7). 2-Octyldibenzo[b,d]thiophene (6) (2 g,
6.7 mmol) and succinic anhydride (742 mg, 7.3 mmol) were added to a round bottom flask, sealed
with a rubber stopper, and placed under a continuous flow of nitrogen gas. To the sealed flask, 14 mL
of 2:1 dichloroethane (DCE)/ nitrobenzene was injected by syringe. The mixture was stirred until a
homogenous solution formed. The flask was then cooled to 0°C by an ice water bath. Upon cooling,
the AlCl3 (1.8 g, 13.4 mmol) was added slowly to the stirring solution over several minutes. Once the
AlCl3 addition was complete, the solution was allowed to stir at room temperature for five hours.
After the fifth hour of room temperature stirring, the solution was warmed to 60°C for 30 minutes.
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The solution was then allowed to cool to room temperature, when it was then poured over HCl
acidified water and washed with DCM 3 x 75 mL. The combined organic layers were dried with
anhydrous MgSO4, then the solution was evaporated under reduced pressure affording a dark oil. The
oil was dissolved in minimal refluxing 2:1 EtOAc:CHCl3 then placed in a freezer overnight. The
resulting solid was recrystallized twice more using the same 2:1 EtOAc/CHCl3 method before
affording an off-white solid (705 mg, 26%). 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 12.17 (s, 1 H),
9.02 (s, 1 H), 8.43 (s, 1 H), 8.15 (d, J = 8.4 Hz, 1 H), 8.05 (d, J = 8.4 Hz, 1 H), 7.96 (d, J = 8.1 Hz, 1
H), 7.42 (d, J = 8.2 Hz, 1 H), 3.45 (t, J = 6.2 Hz, 2 H), 2.78 (t, J = 7.6 Hz, 2 H), 2.61 - 2.69 (m, 2 H),
1.64 - 1.77 (m, 2 H), 1.33 (br. s., 2 H), 1.16 - 1.30 (m, 10 H), 0.84 (t, J = 6.5 Hz, 3 H)13C NMR
(DMSO-d6, 101 MHz): δ (ppm) 198.2, 173.9, 143.9, 139.7, 136.2, 135.1, 135.0, 133.3, 128.4, 125.6,
123.2, 122.8, 122.1, 122.0, 35.1, 33.3, 31.3, 31.3, 28.8, 28.7, 28.7, 28.0, 22.1, 13.9 MS (ESI)
calculated for C24H27O3S- m/z: 395.168, found m/z: 395.1687.
4-(8-Octyldibenzo[b,d]thiophen-2-yl)butanoic acid (8). Zinc powder (310 mg), mercuric chloride
(HgCl2) (33 mg), HCl (15 μL), and DI water (360 μL) were combined in a 50 mL round bottom flask
and stirred for five minutes. After the stirring, the liquid was decanted. To the flask with the
remaining zinc amalgam, DI water (200 μL), HCl (33 μL), toluene (250μL) and 7 (202 mg, 0.5 mmol)
were added. The solution was refluxed for 40 hours. During the reflux, approximately 50 μL of HCl
was added every 6 hours during the day (3 additions). After reflux, the solution is poured over 30 mL
of water acidified by 1.5 mL of HCl. The solution was washed with 3 x 25 mL DCM. The combined
organic washes were dried with MgSO4 and evaporated under reduced pressure with Celite. The
resulting homogenous solid mixture was purified by normal phase flash chromatography using 15%
EtOAc and 85% hexanes with 0.1% acetic acid which elutes byproducts then 65% EtOAc and 35%
hexanes with 0.1% acetic acid. The fractions associated with the product were evaporated under
1
reduced pressure to yield an off-white oil (147 mg, 76%) which eventually solidifies. H NMR
(Chloroform-d, 400 MHz): δ (ppm) 7.94 - 7.97 (m, 2 H), 7.75 (t, J = 7.9 Hz, 2 H), 7.29 (d, J = 8.3 Hz,
2 H), 2.87 (t, J = 7.6 Hz, 2 H), 2.75 - 2.81 (m, 2 H), 2.46 (s, 2 H), 2.04 - 2.13 (m, 2 H), 1.67 - 1.76 (m,
2 H), 1.22 - 1.44 (m, 10 H), 0.86 - 0.92 (m, 3 H)13C NMR (DMSO-d6,101 MHz): δ (ppm) 174.3,
139.0, 138.2, 136.4, 136.1, 135.2, 135.1, 127.7, 127.6, 122.8, 122.6, 121.4, 121.4, 35.1, 34.4, 33.1,
31.3 MS (ESI) calculated for C24H29O2S- m/z: 381.188, found m/z: 381.1895.
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4-(8-Octyl-5,5-dioxidodibenzo[b,d]thiophen-2-yl)butanoic acid (9). 4-(8-Octyldibenzo[b,d]thiophen-
2-yl)butanoic acid (8) (108 mg, 0.28 mmol) was combined with 10 mL of DCM. To this solution,
mCPBA (77%) (292 mg, 1.7 mmol) was added. The solution was stirred overnight for convenience.
After stirring the solution was poured over 50 mL of saturated sodium bicarbonate water. An
additional 20 mL of DCM was added. The organic layer was washed with 4 x 30 mL saturated
sodium bicarbonate water. The organic layer was separated, dried with anhydrous MgSO4, and
filtered. The dried solution was then purified on four separate preparative thin layer chromatography
plates using 1:1 EtOAc: hexanes with 0.1% acetic acid. The silica bands associated with the product
were collected and then filtered with EtOAc with 3% methanol. The filtrate was evaporated under
1
vacuum to afford a white solid (103 mg, 92%). H NMR (400 MHz, Chloroform-d) δ = 8.01 - 7.92
(m, 2 H), 7.75 (t, J = 7.8 Hz, 2 H), 7.30 (s, 2 H), 2.87 (t, J = 7.6 Hz, 2 H), 2.83 - 2.74 (m, 2 H), 2.44 (t,
J = 7.4 Hz, 2 H), 2.11 - 2.06 (m, 2 H), 1.72 (quin, J = 7.4 Hz, 2 H), 1.44 - 1.26 (m, 10 H), 0.93 - 0.84
(m, 3 H)13C NMR (DMSO-d6, 101 MHz): δ (ppm) 174.1, 149.8, 149.0, 135.2, 135.0, 131.3, 131.2,
130.7, 130.7, 122.4, 121.8, 121.7, 35.2, 34.5, 33.0, 31.2, 30.5, 28.7, 28.6, 25.8, 22.0, 13.9 MS (ESI)
calculated for C24H29O4S- m/z: 413.178, found m/z: 413.1794.
Fluorescence quantum yield. Solutions of 4, 9, and naphthalene were made where the absorbance was
0.041 at 250 nm in acetonitrile. The emission spectra were gathered, and the area was integrated using
a function within the instrument software. The fluorescent quantum yield was then calculated using
single point method with Eq. 1.(12)
Fluorescence emission and excitation. The emission and excitation spectra of 4 and 9 were gathered
with the same solutions as used for the quantum yield experiments.
Fluorescence imaging. Sterile glass poly-D-lysine coated #1.5 cover slips (NeuVitro), pre-incubated
with culture medium, were placed in sterile 6-well plates. A single cell suspension of the HeLa
(cervical cancer cell) cell line was prepared at 5 x 104 cells mL-1 in the appropriate medium. Volumes
of 2.0 mL were pipetted into each of the wells containing the coverslips. The 6-well plate was left
overnight at 37.0°C in a 5.0% CO2 incubator. Following the overnight incubation, the plate was
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aspirated dry and then PBS, containing the dye (at 5.0 uM and 0.10% DMSO) in a volume of 2.0 mL,
was gently pipetted onto the cell monolayers attached to the glass cover slips. Cells on the cover slips
were exposed to the dye for approximately 2 hours in the incubator at 37.0°C in a 5.0% CO2. Cells
were washed with PBS and treated with 4.0% paraformaldehyde solution prepared in PBS, incubated
for 30 minutes at room temperature for full fixation. Three washes with 1 mL of PBS were completed
to ensure excess solution was rinsed away. Glass cover slips were removed and mounted on slides
with ProLong Gold Antifade Mountant (ThermoFisher Scientific). The specimen was dried for
several minutes and sealed with clear nail polish before visualized under a microscope.
Fluorescence imaging with co-stain. Sterile glass poly-D-lysine coated #1.5 cover slips (NeuVitro),
pre-incubated with culture medium, were placed in sterile 6-well plates. A single cell suspension of
the HeLa (cervical cancer cells) cell line was prepared at 5 x 104 cells mL-1 in the appropriate
medium. Volumes of 2.0 mL were pipetted into each of the wells containing the coverslips. The 6-
well plate was left overnight at 37.0°C in a 5.0% CO2 incubator. Following the overnight incubation,
the plate was aspirated and PBS, containing the CellMask Orange (ThermoFisher Scientific) (at
1000x dilution with 0.10% DMSO) in a volume of 1.8 mL, was gently pipetted onto the cell
monolayers attached to the glass cover slips. To the CellMask doped PBS 200 μL of 4 or 9 at 50 μM
was added to the respective well. Cells on the cover slips were exposed to the dye for approximately
10 minutes. Cells were washed with PBS and treated with 4.0% paraformaldehyde solution prepared
in PBS, incubated for 30 minutes at room temperature for full fixation. Three washes with 1 mL of
PBS were completed to ensure excess solution was rinsed away. Glass cover slips were removed and
mounted on slides with ProLong Gold Antifade Mountant (ThermoFisher Scientific). The specimen
was dried for several minutes and sealed with clear nail polish before visualized under a microscope.
Imaging capturing. All images were captured with a Leica DM 4000 B microscope with a DFC3000G
camera (Leica Microsystems, Germany) and 63x oil immersion or 40x lenses. HeLa cells were staged
according to the aforementioned methods. Leica LASX Core Software (Leica Microsystems,
Germany) was used to acquire the images.
Image processing and Pearson correlation coefficient calculations. All images were processed using
ImageJ. The Pearson correlation coefficient was calculated using the image of a single cell using an
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ImageJ plugin called Colocalization_Finder by Christophe Laummonerie and Jerome Mutterer of the
Institut de Biologie Moleculaire des Plantes (13).
Antiproliferation studies. HeLa cells were cultured in RPMI (ThermoFisher) supplemented with 10%
FBS (ThermoFisher) and 1% penicillin-streptomycin (ThermoFisher 10000 U/mL). Cells (5000
cells/well in 100 μL) were plated in a 96-well culture plate (Greiner) and incubated for 120 h. Fifty
microliters of the compound dissolved in complete media (final DMSO concentration 1%) at final
concentrations of 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, and 1 nM were added to the appropriate
wells. After incubation of the cells at 37 °C in a humidified atmosphere of 5% CO2 for 120 h, the
cells were stained and incubated with MTS reagent (Promega) for 2 h to determine cell viability. The
absorbance of each well was taken at 490 nm using a multimode plate reader (FlexStation 3,
Molecular Devices, San Diego, CA). GraphPad Prism was used to analyze data to determine the
percent viability. A two-way ANOVA was used to determine if the means of the compounds’ effects
on cell viability were significantly different from each other. Quadruplicate measurements were
performed for each compound.
RESULTS AND DISCUSSION
In previous work, the functionalization of dibenzothiophene (DBT) and its fluorescent analog
DBTOO was posited to direct small molecules into specific cellular components (5). The initial
probing of this hypothesis resulted in the first report of DBTOO based fluorescent microscopy dyes.
The functionalization of DBTOO with sulfonate group and various aryl units produced several
nuclear staining dyes (5). To extend the functionality of DBTOO dyes, lipophilic DBTOO dyes were
desired. The use of butanoic acids with or without the addition of a primary alkyl group flanking the
acid has allowed BODIPY to localize in plasma membranes, and thus, a similar route was pursued for
DBTOO (1).
In an effort to localize DBTOO derivatives in the plasma membrane, two new small molecules
have been produced. The first target molecule did not include a non-polar alkyl tail. The synthesis
involved the addition of an oxobutanoic acid to the second position on the dibenzothiophene (1). This
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was achieved using succinic anhydride in the presence of aluminum trichloride (AlCl3) dissolved in a
2:1 solution of dichloroethane (DCE) and nitrobenzene under an inert atmosphere. Following
sequential recrystallizations in 2:1 ethyl acetate and chloroform, the product 4-(dibenzo[b,d]thiophen-
2-yl)-4-oxobutanoic acid (2) was isolated (11). The product (2) was then reduced to the butanoic acid
form by zinc amalgam to form 4-(dibenzo[b,d]thiophen-2-yl)butanoic acid (3) (11). Carrying out this
reduction with zinc amalgam was not the first choice in reduction techniques due to its toxicity;
however, this process provided the best purification and yields. The reduced product (3) was then
oxidized to the sulfone by six equivalents of mCPBA in DCM. Following the reaction, the solution
was washed with basic water and purified by a normal phase column to afford the final product, 4-
(5,5-dioxidodibenzo[b,d]thiophen-2-yl)butanoic acid (4) as shown in Scheme 1.
>Scheme 1<
The second target molecule included an n-octyl substituent to potentially increase
lipophilicity. The synthesis began with a Grignard reaction with 2-bromodibenzothiophene (5) and
octyl magnesium bromide in dry tetrahydrofuran (THF) with a catalyst. Following workup and
purification with normal phase chromatography,2-octyldibenzo[b,d]thiophene (6) was isolated in
1
good yield (64%) and excellent purity as substantiated by clean H-NMR spectrum(14). The 2-
octyldibenzo[b,d]thiophene (6) was then subjected to the same synthetic pathway that
dibenzothiophene (1) was subjected to in preparing 4. As shown in Scheme 2, the addition of the
oxobutanoic acid to form 4-(8-octyldibenzo[b,d]thiophen-2-yl)-4-oxobutanoic acid (7) was followed
by reduction of the carbonyl to form 4-(8-octyldibenzo[b,d]thiophen-2-yl)butanoic acid (8). Finally,
the oxidation of the sulfide to the sulfone afforded 4-(8-octyl-5,5-dioxidodibenzo[b,d]thiophen-2-
yl)butanoic acid (9) as shown in Scheme 2. Though the same synthetic scheme was used to prepare 4,
the synthesis of 9 produced poorer yields and more problematic purification as evidenced by the use
of preparative thin layer chromatography in lieu of flash chromatography. Nonetheless, the desired
product was isolated in 12% overall yield.
>Scheme 2<
The two dyes, upon their successful synthesis and isolation, had their photophysical data
gathered beginning with their absorbance, which was measured in the visible and ultraviolet region.
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Absorbance spectra were gathered using 0.01 mM solutions of the respective compound in HPLC
grade acetonitrile. The spectra were nearly identical. This was not a surprise as the chromophore was
the same on both molecules; however, 9 has a more substantial absorbance than 4 with a respective
molar absorptivity of 66,000 M-1cm-1 and 51,700 M-1cm-1, respectively. After the absorbance
measurements, the fluorescence emission and excitation spectra was gathered using solutions where
the absorbance of the respective compound was 0.041 at 250 nm in acetonitrile (Figure 1). This was
done to save material as the same solutions were then used to determine the fluorescent quantum
yield, which was not expected to have any impact on the results since no photodecomposition of 4 or
9 was observed in solution. The excitation spectra of 4 and 9 followed the same pattern, though the
two were slightly offset from one another when overlaid with respect to the x-axis.
The emission spectra of 4 and 9 are all but identical, peaking at 359 nm and 357 nm,
respectively. Both emission spectra display a broad undefined shape with no resolved vibronic
features, which was also seen with the first series of DBTOO fluorescent stains (5). There have been
extensive studies of DBT, DBTO, and DBTOO derivatives with the majority displaying no long
lifetime luminescence in solution (15-20). In one exception, 2,8-disubstituion of DBTOO with
phenothiazines, which act as donor moieties, produce a donor-acceptor relationship that lead to a
long-lifetime emission in solution (20).The significant Stokes shift observed for 4 and 9 can suggest a
contribution from longer lifetime luminescence event. However, the luminescence intensity only
decreased 20% when a degassed sample was exposed to air. Since oxygen can completely quench
luminescence involving triplet states, this result suggested the observed luminescence arises from
prompt fluorescence though a contribution from delayed fluorescence or phophorescence cannot be
conclusively excluded.
>Figure 2<
>Table 1<
The dyes, having displayed efficient fluorescence in the visible region, were then imaged
using fluorescent microscopy with HeLa cells. The chosen method, which was reported with the
nuclear DBTOO dyes, was used initially to verify if 4 and 9 were viable dyes (5). This involved
growing the cells on the coverslip and then treating the cells with the respective dye for 2 hours at a
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final concentration of 5 μM. The coverslip was then washed with PBS, treated with 4%
paraformaldehyde in PBS, and left to incubate for 30 minutes at room temperature. The coverslip was
then washed with PBS, mounted to a slide using a mounting gel, and imaged using a Leica
microscope. The replication of the previously reported method afforded good images (Figure 2). The
diffuse emission of the dyes about the entirety of the cell suggests either cytoplasm or plasma
membrane localization. However, this initial round of imaging was done without a co-stain, and thus a
conclusion could not be made. The smaller dye, 4, seen in frames A, B, and C has a stronger emission
from the center of the cell, which suggests the possibility that subcellular organelle membranes may
also be penetrated by the dye. The octyl functionalized dye, 9, possess a more extranuclear emission
indicating that more superficial areas are targeted by this dye as seen in frames D, E, and F of Figure
2. These results show the dyes as functional fluorescent stains and, more importantly, demonstrates
that these dyes may be used for incubation times of up to 2 hours and potentially more.
>Figure 3<
To further optimize staining and understand the localization of the dyes in HeLa cells, a co-
stain was introduced to the cells with the dyes while also utilizing a shorter incubation time. The
modest modification came in two parts. First, the PBS used to replace the media was doped with a
1000x dilution of CellMask Orange, which is a plasma membrane dye, and the incubation time for the
co-stain and dyes was shortened to 10 minutes. These subtle modifications were done to determine if
the dyes, 4 and 9, could be used with short incubation periods and to follow the provided staining
instructions for CellMask Orange. The changes indicated that 4 and 9 localize in the plasma
membrane. CellMask Orange is a known dye which readily localizes in the plasma membrane.
CellMask Orange also does not absorb strongly below 400 nm, and thus, the DAPI filter set used to
image 4 and 9 would not excite the CellMask Orange. The co-stained HeLa cells were imaged by
brightfield and fluorescent techniques where 4 and 9 were viewed through the DAPI filter set and the
CellMask Orange was imaged by a Cy-5 filter set. As seen in frames D and H, both 4 and 9 overlap
well with the respective images taken of CellMask Orange. The overlap was quantified using a
Pearson correlation coefficient calculation with the Colocalization_Finderplugin for ImageJ using a
single cell image cropped from the images included in the Supporting Information (20). The Pearson
correlation coefficient for 4 and 9 with the CellMask Orange were 0.66 and 0.81, respectively, which
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are reasonably strong values affording quantifiable evidence that 4 and 9 localize in the plasma
membrane. The lower value for 4 may be rationalized by referencing the earlier set of images where
the dye likely penetrated intracellular membranes. Keeping this possibility in mind, it is noted that
CellMask Orange does not penetrate well into subcellular membranes with a short incubation time.
Minding those two points affords a reasonable conclusion as to why 4 has a lower Pearson correlation
coefficient compared to 9. The more substantial overlap quantified with regards to 9 may come as a
function the large octyl tail it possesses, which may prevent the dye from readily accessing
intracellular domains. Maintaining 9 primarily in the plasma membrane mimics the effect of
CellMask Orange more closely.
>Figure 4<
Having verified the capacity of 4 and 9 to fluoresce well in cells and localize in the plasma
membrane with a reasonably strong exclusivity, the toxicity of the compounds was determined. The
toxicity of 4 and 9 was determined in HeLa cells. The HeLa cells were treated with concentrations of
the respective dye varying between 1 nm and 100 μM for 72 hours. As seen in Figure 4, neither dye
displayed toxicity at concentrations which would be needed for application by standard imaging
protocols. While the dyes do display toxicity at 100 μM, this level of toxicity is not a concern to most
microscopy related situations.
>Figure 5<
CONCLUSIONS
Two new plasma membrane dyes were designed and synthesized using well-known chemistry.
These dyes displayed efficient fluorescence with fluorescent quantum yields and a meaningful portion
of their respective emission extending into the visible region despite peaking in the UV region, and
thus, they are well visible to both the eye and camera. The photophysics of 4 and 9 was agreeable
with the DAPI filter set of a fluorescent microscope and good images were gathered during both short
and long incubation times. Co-staining with CellMask Orange demonstrated that the dyes localized in
the plasma membranes, and in the case of 4, this may extend into intracellular membranes. Both dyes
possessed reasonable strong Pearson correlation coefficients quantitatively verifying they overlapped
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well with the CellMask Orange which stains the plasma membrane. Thus, this works shows that
DBTOO fluorophores, given the appropriate functionalization, are applicable tools in fluorescent
microscopy.
ACKNOWLEDGEMENTS
This work was supported by the National Science Foundation under CHE-1900417.
SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the
end of the article:
NMR, UV-Vis, and images used for determining Pearson correlation coefficients are available.
REFERENCES
1. Johnson, I. D. (2010) The Molecular Probes handbook. A guide to fluorescent probes and labeling
technologies.Life Technologies Corporation.
2. Specht, E. A., E. Braselmann and A. E. Palmer (2017) A Critical and Comparative Review of
Fluorescent Tools for Live-Cell Imaging. Annu. Rev. Physiol. 79, 93-117.
3. Lavis, L. D. and R. T. Raines (2008) Bright Ideas for Chemical Biology. ACS Chem. Bio. 3, 142-
155.
4. Terai, T. and T. Nagano (2013) Small-molecule fluorophores and fluorescent probes for
bioimaging. Pflügers Archiv - Eur. J. Physiol. 465, 347-359.
5. Petroff II, J. T., K. N. Skubic, C. K. Arnatt and R. D. McCulla (2018) Asymmetric
Dibenzothiophene Sulfones as Fluorescent Nuclear Stains. J. Org. Chem. 83, 14063-14068.
6. Daemen, S., M. A. M. J. van Zandvoort, S. H. Parekh and M. K. C. Hesselink (2016) Microscopy
tools for the investigation of intracellular lipid storage and dynamics. Mol. Metab. 5, 153-163.
This article is protected by copyright. All rights reserved

7. Elle, I. C., L. C. B. Olsen, D. Pultz, S. V. Rødkær and N. J. Færgeman (2010) Something worth
dyeing for: Molecular tools for the dissection of lipid metabolism in Caenorhabditis elegans.
FEBS Lett. 584, 2183-2193.
8. Spangenburg, E. E., S. J. P. Pratt, L. M. Wohlers and R. M. Lovering (2011) Use of BODIPY
(493/503) to visualize intramuscular lipid droplets in skeletal muscle. J. Biomed. Biotechnol.
2011, 598358-598358.
9. Greenspan, P., E. P. Mayer and S. D. Fowler (1985) Nile red: a selective fluorescent stain for
intracellular lipid droplets. J.Cell Bio. 100, 965-973.
10. Kasurinen, J. (1992) A novel fluorescent fatty acid, 5-methyl-BDY-3-dodecanoic acid, is a
potential probe in lipid transport studies by incorporating selectively to lipid classes of BHK
cells. Biochem. Biophys. Res. Commun. 187, 1594-1601.
11. Sidorenko (1984) Synthesis of benzo[b]naphtho[2,3-d]thiophene and 7,8,9,10-
tetrahydrobenzo[b]naphtho[2,3-d]thiophene. Neftekhimiya 24, 3.
12. Lakowicz, J. R. (2007) Principles of Fluorescence Spectroscopy. Springer US.
13. Renz, M. (2013) Fluorescence microscopy—A historical and technical perspective. Cytometry
Part A 83, 767-779.
14. Liu, J., S. Hu, W. Zhao, Q. Zou, W. Luo, W. Yang, J. Peng and Y. Cao (2010) Novel Spectrally
Stable Saturated Blue-Light-Emitting Poly[(fluorene)-co-(dioctyldibenzothiophene-S,S-
dioxide)]s. Macromol. Rapid Commun. 31, 496-501.
15. Gregory, D. D., Z. Wan and W. S. Jenks (1997) Photodeoxygenation of Dibenzothiophene
Sulfoxide:ꢀ Evidence for a Unimolecular S−O Cleavage Mechanism1. J. Am. Chem. Soc. 119,
94-102.
16. Nag, M. and W. S. Jenks (2004) Photochemistry and Photophysics of Halogen-Substituted
Dibenzothiophene Oxides1. J. Org. Chem. 69, 8177-8182.
17. Vos, B. W. and W. S. Jenks (2002) Evidence for a Nonradical Pathway in the Photoracemization
of Aryl Sulfoxides. 1 J. Am. Chem. Soc. 124, 2544-2547.
18. Wan, Z. and W. S. Jenks (1995) Photochemistry and Photophysics of Aromatic Sulfoxides. 2.
Oxenoid Reactivity Observed in the Photolysis of Certain Aromatic Sulfoxides. J. Am. Chem.
Soc. y 117, 2667-2668.
This article is protected by copyright. All rights reserved

19. Dias, F. B., J. Santos, D. R. Graves, P. Data, R. S. Nobuyasu, M. A. Fox, A. S. Batsanov, T.
Palmeira, M. N. Berberan-Santos, M. R. Bryce, A. P. Monkman (2016) The Role of Local
Triplet Excited States and D-A Relative Orientation in Thermally Activated Delayed
Fluorescence: Photophysics and Devices. Adv. Sci. 3, 1600080.
20. Dunn, K. W., M. M. Kamocka and J. H. McDonald (2011) A practical guide to evaluating
colocalization in biological microscopy. Am. J. Physiol. Cell Physiol. 300, C723-C742.
FIGURE CAPTIONS
Figure 1. Dibenzothiophene 5,5-dioxide (DBTOO).
Scheme 1. Synthesis of 4.
Scheme 2. Synthesis of 9.
Figure 2. Fluorescent excitation and emission spectra of 4 and 9.
Figure 3. Fluorescent images of 4 and 9. (A) Bright-field image of cells stained with 4. (B)
Fluorescence image of HeLa cells stained with 4. (C) Merged bright-field and fluorescence images of
HeLa cells stained with 4. (D) Bright-field image of cells stained with 9. (E) Fluorescence image of
HeLa cells stained with 9. (F) Merged bright-field and fluorescence images of HeLa cells stained with
9.
Figure 4. Brightfield and fluorescent images of 4 and 9 co-stained with CellMask Orange. (A) Bright-
field image of cells stained with 4 and CellMask Orange. (B) Fluorescence (DAPI) image of HeLa
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cells stained with 4 and CellMask Orange. (C) Fluorescence (Cy-5) image of HeLa cells stained with
4 and CellMask Orange (D) Merged images of the DAPI and Cy-5 images from staining with 4 and
CellMask Orange. (E) Bright-field image of cells stained with 9. (F) Fluorescence (DAPI) image of
HeLa cells stained with 9 and CellMask. (G) Fluorescence (Cy-5) image of HeLa cells stained with
9and CellMask Orange (H) Merged images of the DAPI and Cy-5 images from staining with 9 and
CellMask Orange.
Figure 5. Metabolic proliferation assay with HeLa cells, 120 h incubation, n = 4.
Table 1. Photophysical data for 4 and 9.
Stokes Shift
Compd. λabs(nm)†,‡ λex (nm)†, § λem (nm)†, ||
(nm)
38
38
log εmax (M-1 cm-1) ¶ Φ†,††
4
9
244
247
244
248
359
357
3.71
3.81
0.4
0.67
†Measured in ACN. ‡Absorbance maxima. §Excitation maxima. ||Emission maxima. ¶Maximum molar
extinction coefficient. ††Fluorescent quantum yield measured by the single point method.
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50
45
40
35
30
25
20
15
10
5
Excitation
4
Emission 4
Excitation
9
Emission 9
0
220
260
300
340
380
420
460
Wavelength (nm)
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O
O
O
S
S
S
OctylMgBr
Zn(Hg)
HCl/ H2O/ Toluene
AlCl3
2:1
DCE: Nitrobenzene
NiCl2(dppp)
THF
O
7
7
Br
5
6
7
O
HO
O
O
S
S
mCPBA
DCM, -30οC
O
O
7
7
8
9
HO
HO