Photostable Voltage-Sensitive Dyes Based on Simple, Solvatofluorochromic, Asymmetric Thiazolothiazoles

Article abstract of DOI:10.1021/jacs.9b08959

A family of asymmetric thiazolo[5,4-d]thiazole (TTz) fluorescent dye sensors has been developed, and their photophysical sensing properties are reported. The π-conjugated, TTz-bridged compounds are synthesized via a single-step, double condensation/oxidation of dithiooxamide and two different aromatic aldehydes: one with strong electron-donating characteristics and one with strong electron-accepting characteristics. The four reported dyes include electron-donating moieties (N,N-dibutylaniline and N,N-diphenylaniline) matched with three different electron-accepting moieties (pyridine, benzoic acid, and carboxaldehyde). The asymmetric TTz derivatives exhibit strong solvatofluorochromism with Stokes shifts between 0.269 and 0.750 eV (2270 and 6050 cm^-1) and transition dipole moments (Δμ = 13-18 D) that are among the highest reported for push-pull dyes. Fluorescence quantum yields are as high as 0.93 in nonpolar solvents, and the fluorescence lifetimes (τ_F) vary from 1.50 to 3.01 ns depending on the solvent polarity. In addition, thermofluorochromic studies and spectrophotometric acid titrations were performed and indicate the possibility of using these dyes as temperature and/or acid sensors. In vitro cell studies indicate good cell membrane localization, negligible cytotoxicity, promising voltage sensitivities, and photostabilities that are 4 times higher than comparable dyes. Their ease of synthesis and purification, remarkable photophysical properties, and chemically sensitive TTz π-bridge make these asymmetric dye derivatives attractive for environmental and biological sensing or similar molecular optoelectronic applications.

Full text of DOI:10.1021/jacs.9b08959

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Article
Photostable Voltage Sensitive Dyes Based on Simple,
Solvatofluorochromic, Asymmetric Thiazolothiazoles
Nickolas Sayresmith, Anand Saminathan, Joshua K. Sailer, Shannon
M. Patberg, Kristin Sandor, Yamuna Krishnan, and Michael G. Walter
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b08959 • Publication Date (Web): 29 Oct 2019
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Photostable Voltage Sensitive Dyes Based on Simple,
Solvatofluorochromic, Asymmetric Thiazolothiazoles
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Nickolas A. Sayresmith,† Anand Saminathan,‡ Joshua K. Sailer,† Shannon M. Patberg,† Kristin
Sandor,† Yamuna Krishnan,‡ and Michael G. Walter*,†
†Department of Chemistry, University of North Carolina at Charlotte, Charlotte, NC, 28223,
‡Department of Chemistry, University of Chicago, and Grossman Institute of Neuroscience,
Quantitative Biology and Human Behavior, University of Chicago, Chicago, IL 60637
*Michael.Walter@uncc.edu
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ABSTRACT: A family of asymmetric thiazolo[5,4-d]thiazole (TTz) fluorescent dye sensors
have been developed and their photophysical sensing properties are reported. The -conjugated,
TTz-bridged compounds are synthesized via a single-step, double condensation/oxidation of
dithiooxamide and two different aromatic aldehydes: one with strong electron donating
characteristics and one with strong electron accepting characteristics. The four reported dyes
include electron donating moieties (N,N dibutylaniline and N,N diphenylaniline) matched with
three different electron accepting moieties (pyridine, benzoic acid, and carboxaldehyde). The
asymmetric TTz derivatives exhibit strong solvatofluorochromism with Stokes shifts between
2270 and 6050 cm-1 and transition dipole moments ( = 13 – 18 D) that are among the highest
reported for push-pull dyes. Fluorescence quantum yields are as high as 0.93 in nonpolar
solvents and the fluorescence lifetimes (F) vary from 1.50 to 3.01 ns depending on the solvent
polarity. In addition, thermofluorochromic studies and spectrophotometric acid titrations were
performed and indicate the possibility of using these dyes as temperature and/or acid sensors. In
vitro cell studies indicate good cell membrane localization, insignificant cytotoxicity, promising
voltage sensitivities, and photostabilities that are 4 times higher than comparable dyes. Their
ease of synthesis and purification, remarkable photophysical properties, and chemically sensitive
TTz -bridge make these asymmetric dye derivatives attractive for environmental and biological
sensing or similar molecular optoelectronic applications.
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KEYWORDS: Thiazolo[5,4-d]thiazole, push-pull, asymmetric, fluorophores, sensitizers,
solvent-dependent Stokes shift, cell membrane voltage sensing.
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INTRODUCTION
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Small molecule fluorophores are attractive molecular tools for environmental sensing
applications and biosensing/bioimaging techniques due to their high microenvironmental
sensitivity, selectivity, and temporal resolution.1 Fluorescent molecules can either turn on/off
their fluorescence or chromically shift their emission through the binding or interaction of
various metal ions,2-5 reactive oxygen species,6 organic toxins,7 or cell organelles/membranes;8-9
and can greatly enhance cell fluorescence microscopic imaging.10 Molecular sensors with large
fluorescence Stokes shifts are advantageous for these applications due to a low overlap between
excitation and emission.8
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A large majority of these molecular derivatives are designed with push-pull (electron
withdrawing and donating) functional groups positioned at either ends of a -conjugated
aromatic core, which facilitates intramolecular charge-transfer (ICT) properties in the excited
state.11-13 Such push-pull dyes typically exhibit strong solvatofluorochromism and high
environmental sensitivity as a result of the spatially separated and strengthened excited-state
dipole.14-15 New push-pull fluorophore discoveries in this field are driven by efforts to enhance
ICT character, and therefore, increase their efficacy for monitoring molecular interactions,
processes, and other events which require high specificity and environmental sensitivity.
Highly fluorescent, thiazolo[5,4-d]thiazole (TTz) chromophores have recently emerged
as an important class of multifunctional heterocyclic compounds.16-19 Molecular materials
containing the fused, bicyclic thiazole backbone have exhibited strong fluorescence and
electrofluorochromism,16 and have been used in a variety of applications such as light-harvesting
dyes in photovoltaics,20-25 redox flow batteries,17 and molecular sensors.19, 26 Additionally,
polymers incorporating the TTz moiety exhibit efficient electronic communication for organic
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field-effect transistors with high free charge carrier mobility and good optoelectronic
tunability.27-30 The rigidity of the TTz structure is especially attractive for molecular
optoelectronics due to their high thermo-oxidative and photochemical stability.25, 30 Surprisingly,
there have been very few reports of asymmetric TTz materials even though there exists a high
demand for tunable, push-pull, small molecular systems that can be incorporated into a
conjugated polymer backbone, or also for use in molecular sensing applications. This is
especially relevant for asymmetric dyes that have shown promising photophysical properties as
light harvesting dyes in small molecule solar cells.23, 31-35 One challenge to the synthesis of
asymmetric TTzs is the fact that the typical reaction pathway for forming the bicyclic
heterocycle requires two, immediately sequential condensation steps, such that the isolation of
the singly condensed intermediate is unfeasible. As a consequence, the introduction of
asymmetry in most previously reported asymmetric TTzs have required the derivatization of an
initially symmetric TTz using post-synthetic, low selectivity, mono-halogenation/mono-
deprotection and Pd cross-coupling reactions.23, 32, 34, 36-38 To emphasize, such routes towards
asymmetric TTzs use multiple synthetic steps, require a similar number of purification
procedures, and often result in low overall yields.
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In an effort to address some of these issues, we have developed a new family of highly
fluorescent, solvatofluorochromic, push-pull TTz compounds that can be accessed using a
simple, single-step reaction (Figure 1a).
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Figure 1. (a) Single step, synthetic reaction to form asymmetric TTz fluorophores and (b) the
four asymmetric TTz compounds explored in this work.
The synthetic strategy begins by heating two different, readily available aromatic aldehyde
precursors with dithiooxamide resulting in one asymmetric and two symmetric TTz
chromophores. Judicious selection of the aromatic aldehyde precursors yields in an amphiphlic
asymmetric TTz whose fluorescence on a column is uniquely distinguishable and whose column
retention is intermediate that of its symmetric counterparts. In this way, silica gel
chromatography gives an easily identifiable asymmetric TTz band (yellow fluorescence) in the
middle of two spatially distant symmetric TTz bands (both with blue fluorescence). Four push-
pull asymmetric TTz compounds were obtained in this manner (Bu2N-TTz-Py, Ph2N-TTz-Py,
Ph2N-TTz-COOH, and Ph2N-TTz-CHO – Figure 1b) containing various electron-donating
groups (dibutylamino, diphenylamino) and electron-withdrawing groups (pyridine, benzoic acid,
and carboxaldehyde). The unique structural features of these chromophores compared to similar
push-pull compounds composed of hydrocarbons is the highly fluorescent, electron deficient,
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heterocyclic TTz bridge connecting the two asymmetric functional groups, whereby the lack of
hydrogens in the TTz bridge allows for enhanced planarity by minimizing any steric effects
typical of hydrocarbons.9, 11-13 The rigid, fused-thiazole, bicyclic aromatic structure provides
thermo-oxidative stability and the increased planarity further enhances the electronic interactions
across the dye.16 This means that compared to typical hydrocarbon-based, fluorescent biological
dyes, the TTz dyes presented herein demonstrate much larger Stokes shifts, enhanced absorption
coefficients, and red-shifted absorption and emission.39 In addition, the thiazolothiazole bicyclic
ring system provides further chemical functionality in a compact, -conjugated bridge. All these
properties make asymmetric TTzs a synthetically simple, photochemically attractive, and novel
class of dyes suitable for environmental and biological sensing.
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EXPERIMENTAL SECTION
Materials and Instrumentation. 4-pyridine carboxaldehyde, 4-(dibutylamino)benzaldehyde, 4-
(diphenylamino)benzaldehyde, terephthalaldehyde, dithiooxamide, and all solvents used for
spectroscopic measurements were purchased from Sigma-Aldrich and used without further
purification. 1H and 13C NMR measurements were obtained with either a JEOL 300 MHz NMR
or a JEOL 500 MHz NMR. High resolution mass spectra were obtained using a ThermoFisher
Scientific MSQ Plus Single Quadrupole Mass Spectrometer. Solution-state UV-Vis spectra were
collected on a Cary 300 UV-Vis spectrophotometer. Time-resolved measurements were taken on
a Jobin Yvon-Spex Fluorolog equipped with a 389 nm diode laser for time-resolved PL decay
measurements. Igor Pro 6.3 software was used to fit PL(t) decay data to single/multiple
exponential decays. Quantum yields were calculated using 9,10-diphenylanthracene as a
reference (quantum yield [F] in cyclohexanes = 0.97).40-41 Density functional theory (DFT)
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calculations were performed with Spartan computational software using B3LYP42-43 density
functional and 6-31G*44 basis set. Temperature studies were conducted using a 10 μM Bu2N-
TTz-Py MeTHF solution placed inside of a Norrell 502 NMR tube and sequentially submerged
in the following liquid N2 baths: octanol (-16 oC), acetonitrile (-41 oC), acetone (-94 oC), pentane
(-131 oC), MeTHF (-136 oC), and liquid N2 itself (-196 oC). The phosphorescence spectrum of
Bu2N-TTz-Py (see S2) was collected similarly at -196 °C by pumping with 389 nm light while
inside a liquid nitrogen bath and then probing the emission within 2 sec of removing from the
bath. Human embryonic kidney cells (HEK 293T) were a kind gift from Prof. Bryan Dickinson,
and Mouse macrophage J774A.1 cells were a kind gift from Prof. Deborah Nelson at the
University of Chicago. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen
Corporation, USA) containing 10% heat inactivated Fetal Bovine Serum (FBS) (Invitrogen
Corporation, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at
37 ºC under 5% CO2.
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Cell labeled spectral measurements. Excitation and emission spectra were collected by
labeling the alveolar macrophage cell line J774.A1 (~ 0.5 x 105 cells) with 1 µM Bu2N-TTz-Py
in phosphate buffered saline (PBS) buffer and 0.1% TritonX-100. Cells were incubated with dye
for 10 min at RT, washed thrice with PBS, and dispersed in a cuvette for fluorescent spectral
measurements (Fluoromax-4, Horiba Scientific, Edison, NJ, USA). Excitation spectra were
collected at 520 nm emission. Emission spectra were collected by exciting at 440 nm.
Electrophysiology. Whole cell voltage clamping measurements of 500 nM Bu2N-TTz-Py
labeled HEK 293T cells were performed with Axopatch 200A amplifier (Molecular Devices),
digitized using an NI-6251 DAQ (National Instruments), and controlled using WinWCP software
(Strathclyde Electrophysiology Software). Patch pipettes were pulled using a Sutter P-97
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Micropipette puller. Patch pipettes with resistances between 5 - 10 MOhm were used in voltage
clamping experiments. The patch pipette was positioned using an MP325 motorized manipulator
(Sutter). Metamorph premier Ver. 7.8.12.0 was linked to an NI-6501 DAQ to enable voltage
triggered image acquisition. Extracellular solution composition was 145 mM NaCl, 20 mM
glucose, 10 mM HEPES, pH 7.4, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2 (310 mOsm); and the
intracellular solution composition was 115 mM potassium gluconate, 10 mM EGTA
tetrapotassium salt, 10 mM HEPES, pH 7.2, 5 mM NaCl, 10 mM KCl, 2 mM ATP disodium salt
(290 mOsm).
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Microscopy. Imaging was carried out on an IX83 inverted microscope (Olympus Corporation of
the Americas, Center Valley, PA, USA) using 100X, 1.4 NA, DIC oil immersion objective
(PLAPON, Olympus) and an Evolve Delta 512 EMCCD camera (Photometrics, USA). Filter
wheel, shutter, and CCD camera were controlled using Metamorph premier Ver 7.8.12.0
(Molecular Devices, LLC, USA). For photostability studies, HEK 293T cells were labeled with
500 nM Bu2N-TTz-Py and illuminated continuously with ~5 W/cm2 power light source,
periodically acquiring images at 1 min interval. Bu2N-TTz-Py channel images were obtained
using 480/20 bandpass excitation filter, 575/40 bandpass emission filter and 89016 dichroic.
VF2.1.Cl channel images were obtained using 500/20 bandpass excitation filter, 535/40 bandpass
emission filter and 69002 dichroic. For CellMaskTM red, images were obtained using the 640/30
bandpass excitation filter, 705/72 bandpass emission filter and 89016 dichroic.
Cellular localization: Time-lapse imaging was performed to probe the membrane localization of
Bu2N-TTz-Py at longer time scale. HEK 293T cells were labelled with 500 nM Bu2N-TTz-Py
and a plasma membrane marker (IX CellMaskTM Red, Thermofisher) for 10 mins in 1X HBSS
(Hank’s balanced salt solution, Thermofisher). Excess unbound dyes were washed with 1X PBS
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and imaged after 30 mins incubation in 1X HBSS. The membrane localized Bu2N-TTz-Py and
CellMaskTM Red were sequentially imaged every 20 mins. Intensity values at plasma membrane
(IPM) were calculated by drawing regions of interest (ROI) around the whole cell and subtracting
intracellular intensity (IIM, ROI drawn within the cell) for Bu2N-TTz-Py and CellMask images,
respectively. The normalized intensity ratio of Bu2N-TTz-Py to CellMaskTM Red, at plasma
membrane (IPM ratio) and intracellular membrane (IIM ratio), were plotted w.r.t. time.
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Cytotoxicity: Cellular toxicity of voltage sensitive dyes was characterized using Annexin V-Cy5
(BioVision) staining of phosphatidylserine that are exposed to outer leaflet of plasma membrane
during apoptosis. HEK 293T cells were labelled with 500 nM of indicated dyes for 15 mins,
followed by incubation with Annexin V-Cy5 for 20 mins in dark and washed, fixed with 4%
paraformaldehyde before imaging. As a positive control, unlabeled HEK cells were incubated at
65°C for 10 mins to induce apoptosis and stained with Annexin V-Cy5 as above. Cytotoxicity
percentage is calculated as number of Annexin V-Cy5 positive cells to total number of nuclear
stained cells. Number of cells quantification were performed using Analyze particle function of
ImageJ program.
2-(N,N-dibutyl-4-aminophenyl)-5-(4-pyridyl) thiazolo[5,4-d]thiazole (Bu2N-TTz-Py): 4-
Pyridinecarboxaldehyde (1.3401 g, 12.511 mmol), dithiooxamide (1.0007 g, 8.3253 mmol), and
4-(dibutylamino)benzaldehyde (2.9108 g, 12.474 mmol) were mixed in 40 mL anhydrous DMF
for 6 h at 120 C. After refrigerating overnight, a brownish-yellow powder precipitate was
collected via vacuum filtration and rinsed with DMSO and water (1.374 g). Using a 1:1
hexanes:chloroform mixture, 31.4 mg of the crude product was purified by silica gel column
chromatography (Silica Flash M60). The eluent was removed under vacuum, thereby yielding a
yellow solid (2.3 mg, 2.9%). 1Η ΝΜR (500 ΜΗz, CD3CN, δ): 8.67 (d, J = 5.05 Ηz, 2Η), 7.84 (d,
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J = 6.20 Ηz, 2Η), 7.79 (d, J = 9.15 Hz, 2H), 6.74 (d, J = 9.90 Hz, 2H). 13C NMR (126 MHz,
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DCM, protonated): 202.19, 197.48, 152.01, 142.06, 142.03. 141.96, 141.94, 129.87, 128.65,
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121.66. UV-Vis λmax (CHCl3, M-1cm-1): 424 nm (ε = 17,700). ESI-MS: calcd for C23H27N4S2 ,
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423.1677; found, 423.1659.
2-(N,N-diphenyl-4-aminophenyl)-5-(4-pyridyl) thiazolo[5,4-d]thiazole (Ph2N-TTz-Py): 4-
Pyridinecarboxaldehyde (0.2577 g, 2.406 mmol), dithiooxamide (0.2519 g, 2.0957 mmol), and
4-(diphenylamino)benzaldehyde (0.8634 g, 3.159 mmol) were mixed in 10 mL anhydrous DMF
for 6 h at 120 C. After sitting overnight, a brownish-yellow precipitate was collected via
vacuum filtration and rinsed with water (0.3201 g). Using chloroform, 20.0 mg of the crude
product was purified by silica gel column chromatography (Silica Flash M60). The eluent was
removed under vacuum, thereby yielding a yellow solid (15.6 mg, 25.8%). 1Η ΝΜR (500 ΜΗz,
d-DMSO, δ): 8.72 (d, J = 5.48 Ηz, 2Η), 7.93 (d, J = 6.40 Ηz, 2Η), 7.89 (d, J = 8.70 Hz, 2H),
7.36 (m, 4H), 7.13 (m, 6H), 6.95 (d, J = 9.15 Hz, 2H). 13C NMR (126 MHz, DCM, protonated):
206.41, 152.86, 151.16, 150.83, 148.69, 148.63, 146.70, 129.61, 127.64, 126.21, 126.21, 125.76,
121.12, 120.28. UV-Vis λmax (CHCl3, M-1cm-1): 424 nm (ε = 46,400). ESI-MS: calcd for
+
C27H19N4S2 , 463.1051; found, 463.0524.
2-(N,N-diphenyl-4-aminophenyl)-5-(4-carboxyphenyl) thiazolo[5,4-d]thiazole (Ph2N-TTz-
COOH): 4-Formylbenzoic acid (0.3774 g, 2.514 mmol), dithiooxamide (0.2002 g, 1.666 mmol),
and 4-(diphenylamino)benzaldehyde (0.6820 g, 2.495 mmol) were mixed in 16 mL anhydrous
DMF for 6 h at 120 C. The reaction mixture was cooled overnight, whereby a brownish-yellow
solid precipitated out of solution. The precipitate was collected via vacuum filtration and rinsed
with water (0.7044 g). Using chloroform, 21.7 mg of the precipitate was purified by silica gel
column chromatography (Silica Flash M60). A yellow solid (6.3 mg, 24.3%) was collected after
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chromatographic separation. 1Η ΝΜR (500 ΜΗz, d-DMSO, δ): 8.10 (d, J = 8.70 Ηz, 2Η), 8.05
(d, J = 8.70 Ηz, 2Η), 7.88 (d, J = 8.70 Hz, 2H), 7.37 (m, 4H), 7.14 (m, 6H), 6.96 (d, J = 8.70 Hz,
2H). 13C NMR (126 MHz, DCM): 203.15, 146.97, 146.79, 144.46, 144.02, 136.09, 132.34,
130.33, 129.59, 127.47, 126.62, 125.66, 124.34, 122.90, 12132. UV-Vis λmax (CHCl3, M-1cm-1):
424 nm (ε = 13,900). ESI-MS: calcd for C29H19N3O2S2, 505.0919; found, 505.0991.
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2-(N,N-diphenyl-4-aminophenyl)-5-(4-formylphenyl) thiazolo[5,4-d]thiazole (Ph2N-TTz-
CHO): Terephthalaldehyde (0.3343 g, 2.49 mmol), dithiooxamide (0.2007 g, 1.67 mmol), and 4-
(Diphenylamino)benzaldehyde (0.6824 g, 2.50 mmol), were refluxed in 10 mL of DMF for 6 h
under aerobic conditions. The reaction mixture was allowed to sit overnight, and the precipitated
product was collected by gravity filtration and rinsed with water (0.5046 g). A portion of the
product (0.1994 g) was then purified by column chromatography on silica gel using
hexanes/dichloromethane (10:1, 4:1) as eluent yielding a yellow solid (17.0 mg, 2.1%). 1H NMR
(300 MHz, DCM, δ): 10.03 (s, 1H), 8.15 (d, J = 8.26 Hz, 2H), 7.95 (d, J = 8.52 Hz, 2H) 7.82 (d,
J = 8.79 Hz, 2H), 7.31 (t, J = 8.79 Hz, 4H), 7.13 (t, J = 7.95 Hz, 6H), 7.05 (d, J = 8.79 Hz, 2H).
13C NMR (126 MHz, DCM): 191.29, 165.94, 152.09, 150.87, 150.56, 146.80, 141.11, 129.13,
137.36, 130.31, 129.59, 127.47, 126.61, 125.66, 124.3304, 121.33. UV-vis λmax (DCM, ε = M-1
+
cm-1): 434 nm (ε = 35,100) ESI-MS: calcd for C29H20N3OS2 : 490.1048; found, 490.1456.
RESULTS AND DISCUSSION
The asymmetric, push-pull TTzs were accessed via one-pot syntheses which include, in
each instance, a donor aromatic aldehyde (D-BzCHO), an acceptor aromatic aldehyde (A-
BzCHO), and dithiooxamide. Initial asymmetric TTz reactions relied on modifying the literature
procedure to accommodate a mixed-aldehyde approach, i.e. using an equivalent molar excess of
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the aromatic aldehydes compared to dithiooxamide (3:2:3). Although accessed simply, the
overall yields (2 - 26%) of the asymmetric TTzs (a-TTzs) were limited by the simultaneous
formation of their symmetric counterparts (generalized within as D2-TTz and A2-TTz). The
yields of Bu2N-TTz-Py and Ph2N-TTz-CHO were significantly lower than Ph2N-TTz-Py and
Ph2N-TTz-COOH. In the case of Ph2N-TTz-CHO, further reaction and polymerization of
carboxaldehyde functionality likely hinders the overall yield. To better understand Bu2N-TTz-Py
synthesis, NMR studies were performed (see Figure S29) and show that the rates of reactivity of
dibutylaminobenzaldehyde (Bu2NBzCHO, the A-BzCHO) and pyridinecarboxaldehyde
(PyBzCHO, the D-BzCHO) differ such that the formation of A2-TTz is favored over a-TTz and
D2-TTz formation. It should be noted that the higher rate of reactivity of the D-BzCHO is
congruent with analogous condensation reactions within the literature.45 Another consequence of
preferential D2-TTz formation is that D-BzCHO may become the limiting reagent for the a-TTz
reaction, thus leading to A2-TTz becoming the secondary product. With these considerations in
mind, the synthesis of Bu2N-TTz-Py was studied by using an excess of the Bu2NBzCHO (1.25 to
1 to 5 mol ratio - PyBzCHO, dithiooxamide, Bu2NBzCHO). NMR of the crude product shows a
TTz ratio of 3:1:1 [Py2-TTz, Bu2N-TTz-Py, (Bu2N)2-TTz], significant amounts unreacted
Bu2NBzCHO starting material, and impurities/side products. To inhibit the formation of side
products, promote complete oxidation,46 and encourage better final yields; the reaction was
modified by reacting under N2, adding dithiooxamide after heating to 120 C, and oxidizing with
DDQ after 4h of reaction time. According to an NMR of the crude product (Figure S30) the
reaction proceeds more cleanly and results in a product ratio of 1.14 to 1 to 0.44 [Py2-TTz,
Bu2N-TTz-Py, (Bu2N)2-TTz].
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The absorption, emission, and fluorescence lifetime characteristics of all four TTz dyes
were recorded in a variety of organic solvents (Figure 2, Table 1). Additionally, HOMO/LUMO
levels of the TTz dyes were determined computationally in Spartan 16 using B3LYP/6-31g*
(Figure 2c, SI). Molar absorptivities of all the TTz dyes ranged from 13,000 to 86,000 cm-1M-1.
In comparison, the molar absorptivities of dipyridyl-TTz (in pyridine) and its alkylated
derivatives (in H2O) range from 32,000 to 46,000 cm-1M-1.16 Interestingly, the peak molar
absorptivities of Ph2N-TTz-Py were two or more times greater than the peak molar absorptivities
of both Bu2N-TTz-Py and Ph2N-TTz-COOH, while those of Ph2N-TTz-CHO were over four-fold
greater than Bu2N-TTz-Py and Ph2N-TTz-COOH in some solvents. TTz dyes containing the
diphenyl substituted donor amine exhibited an increased molar absorptivity compared to the
dibutyl substituted donor amine. However, the peak absorbance of Bu2N-TTz-Py in DCM was
slightly red-shifted as compared to that of Ph2N-TTz-Py (λmax, abs = 429 nm vs λmax, abs = 422 nm
in DCM), which is attributed to the diphenylamine moiety having weaker donor characteristics.
The pyridyl and formylbenzyl acceptor moieties enhance the molar absorptivities of the TTz
dyes relative to the benzoic acid TTz derivative Additionally, Ph2N-TTz-CHO demonstrated the
most red-shifted peak absorbance (λmax, abs = 434 nm in DCM), which is attributed to the 4-
formylbenzyl moiety having stronger acceptor characteristics. Due to the low estimated ground
state dipole moments of the TTz dyes ( = 6 – 8 D), their wavelengths of absorbance remained
relatively solvent independent (st. dev. = ±11 nm).47
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The TTz dyes exhibit high quantum yields (QYs) in nonpolar solvents; however, as the
polarity of the solvent increases, the emission intensities and, correspondingly, the QYs decrease
(e.g. for Bu2N-TTz-Py: hex = 0.93, MeOH = 0.04). The decrease in the QY is common for dyes
with strong excited-state ICT character.12, 48-49 In spite of having low QYs in polar solvents, the
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TTz dyes could be expected to perform well in applications where they are localized to nonpolar
environments (e.g. cell membranes); whereby their fluorescence would increase upon
localization and any unlocalized dyes would contribute little background noise.47
TCSPC measurements of the TTz dyes show a general upward trend for fluorescent
lifetimes as the polarity increases in aprotic solvents – from 1.50 – 1.89 ns in nonpolar solvents
to ~3.00 ns in polar aprotic solvents (Table 1). The fluorescence lifetimes in protic solvents have
the opposite trend; that is, a decrease in the fluorescence lifetime as the polarity increases (e.g.
for Ph2N-TTz-COOH: τf, iPrOH = 2.40 ns, τf, MeOH = 1.80 ns). The trend reversal in the
fluorescence lifetime can most likely be attributed to the presence of hydrogen bonding in protic
solvents. Due mostly to large changes in the QYs, but also to increasing fluorescent lifetimes in
more polar aprotic solvents, the radiative rates (kr) decrease relatively significantly with
increasing solvent polarity. At the same time, the non-radiative rates of each TTz generally
increases as solvent polarity increases. As with the asymmetric TTz dyes, it has been shown that
neutral red (NR), another ICT dye, shows a similar trend.50 Regardless, the fact that the
fluorescence lifetimes are solvent dependent opens up the possibility of using these TTz dyes for
measuring micelle formation and viscosity in living cells using fluorescence lifetime imaging
(FLIM).1, 51
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Figure 2. (a) Normalized UV-vis absorbance and solvent-dependent fluorescence, (b) solvent-
dependent time-resolved fluorescence lifetimes, and (c) HOMO/LUMO molecular orbital
configurations for Bu2N-TTz-Py.
The presence of dual fluorescence and its dependence on solvent polarity is a well-
documented phenomenon in many singly bonded D-A molecules with excited states that have
highly dipolar quinoidal structures.52 The higher frequency peak in nonpolar solvents can mostly
be attributed to the locally excited (LE) state, whereas the lower frequency peak can mostly be
attributed to the ICT state.53 As is typically seen in similar D-A systems, it is suspected that the
ICT state in the asymmetric TTz dye system is stabilized by the solvating effect of the polar
solvent, thus causing the emission intensities of the dyes to broaden, diminish, and shift
bathochromically with increasing solvent polarity. Another consequence of having electronically
separated LE and ICT states is that the fluorescence lifetimes can increase in high-polar solvents
even though the QYs are decreasing likewise; whereas, in typical non-ICT systems the
fluorescence lifetimes and QYs are directly related.47, 50
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Table 1. Optical Properties of TTz Dyes in Various Solvents
Non-
radiative
rate
5
6
7
8
Fluorescence
Lifetime
(ns)
Radiative
rate
Polarizability
Factor
Abs
(nm)
Abs
(eV)
Ext. coeff.
(M·cm)-1
Em
(nm)
Em
(eV)
Stokes
Shift
(sec-1)
(sec-1)
SS
(eV)
Δ푓
*
dye
solvent
λmax
Eex
ɛ
λmax
Eem
τF
kr
knr
F
9
Hex
DCM
-0.00170
0.217
410
429
430
425
424
3.02
2.89
2.88
2.92
2.93
22000
13000
18000
20000
21000
450
515
532
557
565
2.76
2.41
2.33
2.23
2.20
0.27
0.48
0.55
0.69
0.73
0.93
0.35
0.45
0.17
0.04
1.50
2.47
2.26
3.06
1.23
6.2E+08
1.4E+08
2.0E+08
5.4E+07
3.1E+07
4.4E+07
2.6E+08
2.4E+08
2.7E+08
7.8E+08
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Bu2N
-TTz-
Py
CHCl3
MeCN
MeOH
0.148
0.305
0.309
Tol
DCM
0.0132
0.217
0.148
0.199
0.284
419
422
424
415
415
2.96
2.94
2.93
2.99
2.99
36000
34000
46000
48000
35000
482
536
518
518
554
2.57
2.31
2.39
2.39
2.24
0.39
0.63
0.53
0.59
0.75
0.93
0.37
0.54
0.55
0.17
1.80
3.16
2.60
2.80
2.70
5.2E+08
1.2E+08
2.1E+08
2.0E+08
6.3E+07
3.9E+07
2.0E+08
1.8E+08
1.6E+08
3.1E+08
Ph2N
-TTz-
Py
CHCl3
EtOAc
Acetone
Tol
0.0132
0.217
0.148
0.272
0.309
424
424
425
417
414
2.93
2.93
2.92
2.97
3.00
22000
21000
14000
16000
17000
487
523
548
522
535
2.55
2.37
2.26
2.38
2.32
0.38
0.55
0.66
0.60
0.68
0.78
0.33
0.55
0.37
0.11
1.70
2.90
2.20
2.40
1.80
4.6E+08
1.1E+08
2.5E+08
1.5E+08
6.1E+07
1.3E+08
2.3E+08
2.0E+08
2.6E+08
4.9E+08
Ph2N
-TTz-
DCM
CHCl3
iPrOH
MeOH
COO
H
Tol
ClBz
0.0133
0.143
0.186
0.200
0.217
425
436
440
428
434
2.92
2.84
2.82
2.90
2.86
80230
18024
37000
86081
35100
501
552
556
548
560
2.48
2.37
2.26
2.26
2.21
0.44
0.55
0.66
0.63
0.64
0.72
0.33
0.18
0.36
0.13
1.89
2.54
2.84
3.09
2.40
3.8E+08
1.3E+08
6.3E+07
4.2E+07
1.5E+08
1.5E+08
2.6E+08
2.9E+08
7.5E+07
1.0E+09
Ph2N
-TTz-
CHO
Cl2Bz
EtOAc
DCM
*
Δ푓, 푠푒푒 퐸푞. 1)
Solvents were chosen to span a wide range of polarizability factors (
. For direct comparisons, similar solvents
were used when possible; however, solubility differences between the compounds prohibited complete standardization in this
Δ푓
regard. To compensate, alternative solvents were selected based on solubility and
were not soluble in hexanes ( ), so toluene (
proximity. E.g. the diphenylamino TTzs
) was chosen instead.
Δ푓 = ― 0.0017 Δ푓 = 0.013
As shown in Figure 2 and listed in Tabe 1, the TTz dyes exhibit strong
solvatofluorochromism – of which Bu2N-TTz-Py shows the largest Stokes shift of the four TTz
dyes (0.73 eV). The calculated HOMO/LUMO levels indicate that the strong
solvatofluorochromism arises from an ICT mechanism, whereby the ground state has significant
electron localization on the donor side of the molecule whereas the electron density shifts to the
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acceptor moiety in the excited state. To more directly quantify the strong ICT character of the
TTz dyes, the excited-state dipole moments were calculated using the Lippert-Mataga equation
(Eq 1):
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2
∗
휂2 ― 1
2휂2 + 1
(
)
휇 ― 휇
휖 ― 1
2휀 + 1
휈푎 ― 휈푓 =
Δ푓 + 푐표푛푠푡. ; Δ푓 =
―
(
)
4πϵ0ℎ푐푎3
where and 푓 are the wavenumbers of the absorption and emission peaks in cm-1, respectively;
휈푎
휈
휇 ∗
permittivity, h is Planck’s constant, c is the speed of light, a is the Onsager cavity radius,
휇
ϵ
and are the excited state and ground state dipoles, respectively; 0 is the vacuum
is
Δ푓
the orientation polarizability, is the relative permittivity, and is the refractive index. The
휖
휂
dipoles were calculated using Spartan 2016 software, and the Onsager cavity radius was
calculated using Gaussian ‘09 software.
Figure 3. (a) Lippert-Mataga plots of Bu2N-TTz-Py and (b) Ph2N-TTz-COOH.
Figure 3 shows the Stokes shift of Bu N-TTz-Py and Ph N-TTz-COOH versus . From
훥푓
2
2
the slopes of the Lippert-Mataga plots, the change between the dipole moments of the ground
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and excited states can be determined. Even though the Lippert-Mataga equation assumes no
specific solvent-solute interactions (e.g. hydrogen bonding) and ignores solute polarizability,
high degrees of linearity are apparent, and therefore, the estimations of the excited state dipoles
are regarded as reliable. Bu2N-TTz-Py, in particular, shows a near unity correlation with Lippert-
Mataga theory, thus suggesting minimal specific solvent-solute interactions and/or relatively
rapid vibrational relaxation as compared to solvent relaxation.11, 54-55 Perhaps owing to the
minimal solvent interactions and rapid vibrational relaxation, the estimated changes in dipole
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moments ( ) are remarkably large (Table 2). Bu N-TTz-Py, for example, has a calculated
∆휇
2
∗
15.9 D and a
24.3 D. Compared to similar small-molecule, push-pull dyes, the
∆휇 =
휇 =
asymmetric TTz dyes have some of the largest ever reported s – twice that of Prodan and
∆휇
comparable to FR0 and 7AHC.12, 56 Therefore, it is expected that these asymmetric TTzs dyes
would be well suited to give high sensitivity in sensing applications whereby small changes in
the polarity of the environment can lead to large differences in the wavelengths of fluorescence.
Table 2. Ground and Excited State Dipole Moments
Compound
Onsager Cavity Radius
Ground State Dipole
Excited State Dipole
Change in Dipole
∗
a (Å)a
a
흁
(D)b
b
흁
(D)
∆흁
(D)
Bu2N-TTz-Py
6.17
6.11
6.16
6.22
8.34
6.06
8.36
6.89
24.3
20.8
21.3
24.9
15.9
14.6
13.0
18.0
Ph2N-TTz-Py
Ph2N-TTz-COOH
Ph2N-TTz-CHO
a Calculated using DFT B3LYP/6-31G(d) with tight SCF, finegrid integral, and volume keyword
b Semi-empirically calculated using the Lippert-Mataga Equation
In an extension of sensing the surrounding polarity, the dependence of the wavelength of
emission as a function of temperature and pH were also explored (for pH studies see SI). For
these efforts, Bu2N-TTz-Py was chosen as a focal representative for our family of TTzs since it
had the highest Stokes shift and otherwise similar photophysical characteristics. For the
temperature studies, a 10 µM MeTHF solution of Bu2N-TTz-Py was created and the emission of
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the solution was monitored from -196 – 60 °C (see Figure S2 for phosphorescence). MeTHF
was chosen for its wide liquid temperature window and ability to form a glass upon freezing;
most other solvents crystallize upon solidification and inhibit fluorescence. The low temperature
studies were achieved using various liquid N2 cooling baths (see Experimental). Due to the
condensation of atmospheric moisture during low-temperature testing, only the normalized
emission intensities are reported. As seen in Figure 4, Bu2N-TTz-Py exhibits
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thermofluorochromism, whereby the wavelength of emission shifts bathochromically as
temperature decreases (from 519 nm at 60 °C to 559 nm at -131 °C). At temperatures near the
freezing point of MeTHF and below, however, the wavelength of emission rapidly blue-shifts
from 559 to 544 to 503 nm at -131, -136, and -196 °C, respectively. Because the solvent polarity
is dependent on the temperature of the solvent, the presence of thermofluorochromism could be
fully expected, i.e. it is a direct analogue of solvatofluorochromism.57 As a corollary, any solvent
which also dissolves Bu2N-TTz-Py will show a similar temperature affect; however, it is
expected that the operable temperature range, sensitivity, and linearity would change
correspondingly. Whereas as most dye systems demonstrate a hypsochromic shift upon
temperature reduction, Bu2N-TTz-Py demonstrates the opposite trend. General intuition might
infer that the direct relationship between viscosity and temperature would likewise lead to faster
solvation relaxation at higher temperatures and consequently red-shift the emission spectra.
Higher temperatures, however, can also prevent the alignment of solvent dipoles, thus leading to
a blue shift in the emission spectra as temperature increases – such as is seen for Bu2N-TTz-Py
and, previously, for Laurdan.47, 58 Furthermore, the hypsochromic shift of the emission that was
observed upon freezing is a well-known phenomenon, and can best be attributed to the complete
inhibition of solvent relaxation effects upon freezing.47 Nevertheless, to visualize the temperature
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dependence of the emission wavelength, a temperature-wavelength correlation profile for Bu2N-
TTz-Py in MeTHF was created (Figure 4). It was observed that the wavelength of emission
shows a strong linear correlation with the solution temperature while inside the temperature
range in which MeTHF is liquid. Additionally, the TTz/MeTHF solution showed relatively high
temperature sensitivity (-0.21 nm °C-1), thus making it suitable for temperature sensing
applications.59-62
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Figure 4. (a) Normalized emission intensity spectra of Bu2N-TTz-Py and (b) temperature-
wavelength correlation profile of Bu2N-TTz-Py in MeTHF when T > -136 °C (blue) and T ≤ -
136 °C (orange).
Having characterized the sensing capabilities of the asymmetric TTz dyes in solution,
their efficacy as biologically relevant sensors was then explored via in vitro cell studies. Bu2N-
TTz-Py was selectively chosen based on its expected favorability toward membrane localization
given its molecular structure (hydrophilic pyridyl head, hydrophobic dibutylamino tail). As
shown in Figures 5a/5b, when Bu2N-TTz-Py is applied to HEK 293T cells, the dye localizes to
cell membranes, displaying peak excitation/emission wavelengths at 423 and 483 nm,
respectively (Figure 5c). Also noteworthy, the wavelength of peak emission for Bu2N-TTz-Py in
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a solution of PBS with 0.1% Triton-X100 (518 nm) blue-shifts by 34 nm upon localizing to a cell
membrane. A 34 nm blue shift is equivalent to the solvent polarity differential between DCM
and toluene, thus further indicating that Bu2N-TTz-Py intercalates between the phospholipid
bilayer. Additionally, Bu2N-TTz-Py has both poor solubility and low QY in aqueous
environments resulting in low background fluorescence, which is visually apparent by the high
amount of contrast between the cell membrane and its surrounding environment (Figure 5b).
More conclusively, Figures 5d/5e show the time lapse imaging and quantification of localization
of Bu2N-TTz-Py in HEK 293T cells (6 independent experiments). Normalized against the
fluorescence intensity of CellMaskTM red, a plasma membrane marker, the fluorescence
intensities of the plasma membranes stained with Bu2N-TTz-Py shows a 2.5% decrease after 50
min (26% decrease after 110 min). Given the brevity of the acquisition time (6 acquisitions per
cell, 200 ms exposure per acquisition), the overall decrease of the plasma membrane
fluorescence intensities of Bu2N-TTz-Py is most likely not caused by photodegradation.
Furthermore, the decrease of fluorescence intensities is not caused by dye internalization in any
significant capacity since the normalized intensities of intracellular membranes stained with
Bu2N-TTz-Py indicate negligible TTz internalization (< 1% over 50 min, 5% over 130 min). It is
therefore concluded that the majority of the fluorescence reduction after 50 min is caused by
extracellular leaching.
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Figure 5. In vitro cellular characterization of Bu2N-TTz-Py. (a, b) DIC and fluorescent images
of HEK 293T cells labelled with 500 nM Bu2N-TTz-Py in PBS buffer containing 0.01% Pluronic
F127. (c) Normalized excitiation/emission spectra of 1 μM Bu2N-TTz-Py in PBS buffer with
0.1% TritonX-100 before and after loading into ~0.5 x 105 J774.A1 cells. Exctitation spectra (- -)
were collected at 520 nm and emission spectra (-) were collected with 440 nm exctiation. (d)
Time lapse imaging of HEK 293T cells show partial internalization of Bu2N-TTz-Py, but no
internalization of CellMask stain after 130 min. (e) Quantification of intracellular membrane (red
trace, circles) and plasma membrane (black trace, squares) localization of Bu2N-TTz-Py over
time. Error bars represent S.E.M. of 6 independent experiments. Scale bar = 10 µm.
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Figure 6. (a) Plot of fractional change in fluorescence (ΔF/F) vs clamped membrane potential of
HEK 293T cells labelled with 500 nM Bu2N-Py-TTz and representative pseudo-color images
clamped at -100, 0, and +100 mV (shown by white arrow). Inset: Voltage clamped cell. (b)
Photostability of Bu2N-TTz-Py and VF2.1.Cl in HEK 293T cells (illuminated using λ = 440 nm
at 5 W cm-2) and their representative psuedo-color images under continous illumination at
wavelength of maximum excitation. Error bars a/b represent the standard error of mean (S.E.M.)
for 3 independent experiments, n = 12 total cells. Scale bar = 10 μm. (c) Annexin V-cy5 staining
of HEK 293T cells incubated with DMSO (Veh.), VF2.1.Cl, Bu2N-TTz-Py. Apoptosis for
control (incubated cells 65 °C, 10 min). Percentages of cytotoxicity were calculated as number of
100 X Cy5 positive cells per number of nuclei stained. Error bars represent S.E.M. of three
independent experiments, n = 250 cells. Scale bars = 10 µm.
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Having established its environmental sensitivity and proclivity for membrane
localization, it was hypothesized that Bu2N-TTz-Py could orient itself favorably within cell
membranes and sense voltage differentials across the membrane. To test the membrane voltage
sensitivity of Bu2N-TTz-Py, whole-cell patch clamp electrophysiology was performed on HEK
293T cells labeled with 500 nM Bu2N-TTz-Py. As seen in Figure 6a, cells were held at -60 mV
and stepped through 10 mV increments whereby depolarizing or hyperpolarizing voltage
potentials resulted in either a florescence increase or decrease, respectively. Tests concluded that
Bu2N-TTz-Py has a voltage sensitivity of approximately 10% ΔF/F per 100 mV. For context,
early generation VSDs, such as RH-421, ANNINE-6, and VF2.1.Cl, display maximum voltage
sensitivities of 10% – 25% ΔF/F per 100 mV.63-64 In other words, Bu2N-TTz-Py shows a
comparatively modest voltage sensitivity relative to comparable VSDs in the literature. It should
also be noted that the voltage sensitivities, signal-to-noise, and linearity of VSDs with ICT
character (e.g. Bu2N-TTz-Py) can vary significantly depending on both the wavelength of
excitation and probed emission.65
Encouraged by the promising voltage sensitivity data, the photostability of Bu2N-TTz-Py
was also tested and found to be much improved over the photostability of VF2.1.Cl (Figure 5b).
Whereas VF2.1.Cl had a bleaching half-life of approximately 7.5 minutes under intense
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illumination (I = 5 W cm-2, ɛ = 22,300 cm-1 M-1 @ 440 nm LED10), the fluorescence intensity of
Bu2N-TTz-Py decayed by less than 10% in the same amount of time under identical conditions (ɛ
= 15,000 – 20,000 cm-1 M-1 @ 440 nm LED). The improved photostability of Bu2N-TTz-Py over
that of VF2.1.Cl can best be attributed to the structural difference between both their head groups
and bridging units. It is well known that fluorescein has low photostability and, in the case of
VF2.1.Cl, it has been shown that derivitization of its fluorescein head group can drastically
improve the photostability.66-67 Additionally, p-phenylenevinylene (PPV), i.e. the bridging unit in
VF2.1.Cl, has been shown to have photostability issues as well. 62-64 TTz, on the other hand, has
excellent thermo-oxidative and photochemical stability. 25, 29 Furthermore, TTz has the added
advantage of requiring only a single condensation step, whereas most other VSDs (including
VF2.1.Cl) require multiple synthetic steps. However, the overall yields of the asymmetric TTz
dyes and other VSDs remain similar.
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Lastly, the cellular toxicities of Bu2N-TTz-Py and VF2.1.Cl were characterized using
Annexin V-Cy5 staining of phosphatidylserine. HEK 293T cells were labelled with Bu2N-TTz-
Py or VF2.1.Cl for 15 min, incubated with Annexin V-Cy5 for 20 min in the dark, washed, and
fixed with 4% paraformaldehyde before imaging. As a positive control, apoptosis was induced
via high-temperature incubation (65 °C, 10 min). As shown in Figure 5c, the treatments of
Bu2N-TTz-Py or VF2.1.Cl to HEK 293T cells both show insignificant cytotoxicity.
CONCLUSION
In conclusion, we have developed a family of asymmetric TTz dyes which exhibit
remarkable solvatofluorochromism as a consequence of the unique structural features provided
by the TTz core, namely its enhanced rigidity and planarity in which typical conjugated,
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hydrocarbon bridges do not confer. Spectroscopic studies of these TTz dyes reveal large
absorption coefficients, high quantum yields in nonpolar environments, significant Stokes shifts,
and red-shifted absorption/emission peaks across a wide solvent range. Lippert-Mataga plots
indicate that these TTz dyes have some of the largest dipole moments ever reported ( = 13 –
18 D) - double that of Prodan and comparable to FR0 and 7AHC. Additionally, we have showed
that the remarkable photophysical properties of the fused, bicyclic thiazolothiazole -bridge
make them ideal for polarity and/or temperature sensing applications. Furthermore,
electrophysiological, in vitro cell studies indicate promising voltage sensitivities, negligible
cytotoxicity, and photostabilities 4 times higher than that of VF2.1.Cl. Thus, the strong ICT
character and remarkable photophysical properties of these new asymmetric TTz dyes make
them attractive for a wide range of sensing applications.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Optical band gaps, absorption and emission spectra, phosphorescence spectrum, DFT,
TD-DFT, electrostatic potential maps, and all spectrophotometric titration information.
AUTHOR INFORMATION
Corresponding Author
*Michael.Walter@uncc.edu
ORCID
Michael G. Walter: 0000-0002-9724-265X
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Notes
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The authors declare no competing financial interests.
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ACKNOWLEDGMENTS
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This research was funded by the Department of Chemistry at the University of North Carolina at
Charlotte, the B.S. Chemistry Program, and the Nanoscale Science Ph.D. program. MGW also
acknowledges ACS Project SEED, Charlotte Research Scholars, and NSF-REU/DOD-ASSURE
(CHE-1460867). Special thanks for research assistance from Brandon Miller, Jackson Mower,
Jon Palmer, Mirna Peralta, Sashank Sabbineni, Natalie Herr, and Andrew Hyunn.
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