A comprehensive spectral, photophysical and electrochemical study of synthetic water-soluble acridones. A new class of pH and polarity sensitive fluorescent probes

Article abstract of DOI:10.1016/j.dyepig.2019.03.028

A comprehensive spectroscopic, photophysical and electrochemical investigation of N-alkylacridones, their newly synthesised chlorosulfonyl and water-soluble sulfonic acid derivatives has been undertaken in solution at room temperature. The study includes absorption and emission spectra together with quantitative measurements of the deactivation of the first excited singlet state, from which the rate constants for all the decay processes has been obtained. A comparison on the gradual change of the electronic spectral and photophysical properties with the degree of substitution is considered. In addition the electrochemical behavior of the water-soluble acridones at different pH values was obtained by cyclic and differential pulse voltammetry. The oxidation mechanism of disulfonated acridone is found to occur in one consecutive and irreversible charge transfer pH-dependent reaction. The water-soluble acridones were found to display long lifetime values, dependent on the pH and solvent polarity. This opens a door for its use as selective probes for different cellular environment and targets.

Full text of DOI:10.1016/j.dyepig.2019.03.028

Accepted Manuscript
A comprehensive spectral, photophysical and electrochemistry study of synthetic
water-soluble acridones. A new class of pH and polarity sensitive fluorescent probes
Ricardo C. Pereira, Ana Dora R. Pontinha, Marta Piñeiro, J. Sérgio Seixas de Melo
PII:
S0143-7208(18)32828-6
https://doi.org/10.1016/j.dyepig.2019.03.028
DYPI 7417
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 22 December 2018
Revised Date: 20 February 2019
Accepted Date: 12 March 2019
Please cite this article as: Pereira RC, Pontinha ADR, Piñeiro M, Seixas de Melo JSé, A comprehensive
spectral, photophysical and electrochemistry study of synthetic water-soluble acridones. A new class of
pH and polarity sensitive fluorescent probes, Dyes and Pigments (2019), doi: https://doi.org/10.1016/
j.dyepig.2019.03.028.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Manuscript for
Dyes & Pigments
A comprehensive spectral, photophysical and electrochemistry study of
synthetic water-soluble acridones. A new class of pH and polarity
sensitive Fluorescent Probes
Ricardo C. Pereiraa, Ana Dora R. Pontinhaa, Marta Piñeiroa, J. Sérgio Seixas
de Meloa*
aCQC, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535
______________________________________________________________________
ABSTRACT
A comprehensive spectroscopic, photophysical and electrochemical investigation of
N-alkylacridones, their newly synthesised chlorosulfonyl and water-soluble sulfonic acid
derivatives has been undertaken in solution at room temperature. The study includes
absorption and emission spectra together with quantitative measurements of the
deactivation of the first excited singlet state, from which the rate constants for all the
decay processes has been obtained. A comparison on the gradual change of the electronic
spectral and photophysical properties with the degree of substitution is considered. In
addition the electrochemical behaviour of the water-soluble acridones at different pH
values was obtained by cyclic and differential pulse voltammetry. The oxidation
mechanism of disulfonated acridone is found to occur in one consecutive and irreversible
charge transfer pH-dependent reaction. The water-soluble acridones were found to display
long lifetime values, dependent on the pH and solvent polarity. This opens a door for its
use as selective probes for different cellular environment and targets.
______________________________________________________________________
Keywords: Acridones • Sulfonated Acridones • Photophysics • Electrochemistry •
Fluorescent probes
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INTRODUCTION
Nowadays, there is a considerable interest in the development of fluorescence
lifetime-based biological probes for assays in biophysics, clinical diagnostics, DNA
analysis, and drug delivery. Unlike steady state fluorescence (based on emission variation
of intensities), time-resolved fluorescence provides a signal (and value) that can be
discriminate against many experimental artefacts resulting from steady-state
measurements. Generally time-resolved fluorescence is independent of the probe
concentration and volume, it remains unaffected by auto-fluorescence of biological
samples or light scattering, and thus leads to more robust assays. Considering that
fluorescent cellular (intrinsically) components typically have fluorescence lifetime shorter
than 5 ns and that the more widely used (extrinsically) fluorophores such as fluorescein,
rhodamine, coumarins or BODIPY´s[1-6] display lifetimes that are shorter than 10 ns,
there is interest to develop fluorophores with long fluorescence lifetimes (with values of
more than 10 ns), sensible to change in the environment and soluble in biological
medium[7, 8].
Acridones, a subclass of acridines with a core structure consisting of 9(10H)-
acridone, Scheme 1, are a group of compounds with applications in various fields, such as
medicinal chemistry[9] namely as anti-viral[10, 11], anti-bacterial[12], anti-parasite, anti-
fungi drugs[13], in single nucleotide polymorphism detection in DNA variation[14] and
for the treatment of the malaria [15-17] and cancer[18-20].
O
N
H
Scheme 1: Structure of Acridone
Acridone (AC) is also a well-known highly fluorescent fluorophore, with more than
50% of the quant loss made through the radiative channel [21, 22] and an emission (color)
that is sensitive to its environment, which are both desirable attributes relevant to
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designing media-sensitive chromophores for biolabeling purposes. Therefore, acridones
and their derivatives have been used as chemosensors and fluorescent markers [23-29].
The spectroscopic characterization of 10-alkyl-9(10H)-acridone derivatives show
the sensitivity of the absorption and emission maxima to the polarity of the solvent and the
involvement of an electron transfer process in the fluorescence quenching [25].
Additionally, spectro-electrochemical experiments showed that the compounds oxidize
irreversibly at low oxidation potential [30]. To date, the power conversion efficiency of
solar cells using small molecules as donors, such as acridones, have exceeded 8%, making
these materials ideal candidates for the solution processed Dye-Sensitized Solar Cells
(DSSCs). [31, 32]
In our effort to obtain new probes presenting long-term stability for different
applications including electrolytes for redox-flow batteries, sensing of oxygen, etc., we
have made versatile acridones (and its alkyl derivatives) soluble in water with the
introduction of the sulfonic group.[33, 34] In the present study, the spectral, photophysical
and redox properties of the new water-soluble acridone derivatives were investigated in
DMSO and water (at different pH values).
EXPERIMENTAL SECTION
Material and methods
Acridone (Aldrich), chlorosulphonic acid (Aldrich) and dimethyl sulfoxide (DMSO)
of spectroscopic grade were used without further purification. Ultra-pure water at pH=5.4,
was purified using a Direct Q3 Merck Millipor equipment. Ethyl Acetate was purified in
reflux with potassium carbonate during 3 hours, distilled and then stored with molecular
sieves (4 Å). When required, thin layer chromatography (TLC) silica plates 60 F250
(Merck or Fluka) were used. Absorption spectra were recorded on a Cary5000 UV-Vis-
NIR spectrophotometer. Fluorescence spectra were recorded in a Horiba-Jobin-Yvon-Spex
Fluorolog 3-2.2. spectrofluorimeter. The fluorescence quantum yields were determined
using quinine bisulfate as standard (φF = 0.546 in a 0.5 M H2SO4 aqueous solution).[35]
The molar absorptivity constants (ɛSS, Table 1) were obtained by the adjustment of a slope
on the plot of the absorption vs. concentration with correlation values above 0.99, obtained
by preparing 4 solutions with concentrations ranging from 8x10-5 to 3x10-5 M.
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The fluorescence decay times were obtained by the TCSPC (Time-correlated Single
Photon Counting) technique with nanosecond resolution in an equipment described
elsewhere[6, 36], and analysed using the method of modulating functions implemented by
G. Striker[37]. Microwave assisted synthesis was carried out on a CEM Discover S-Class
equipment with constant monitorization of temperature, pressure and potency.
NMR spectra were recorded on a Bruker Avance 400 spectrometer, operating at
400.13 MHz for 1H NMR and 100.62 MHz for 13C NMR. Chemical shifts (δ) are reported
in ppm relatively to tetramethylsilane (TMS). High-resolution mass spectrometry analysis
was carried out on a Bruker Microtof apparatus, equipped with selective ESI-TOF
detector. Melting points were determined using a Melting Point Device Falc R132467
(open capillary method). Infrared spectra were recorded with an Agilent Technologies
Carey 630 FTIR spectrometer with ATR.
General procedure for the synthesis of N-alkyl-9(10H)-Acridone derivatives.
The synthesis of the different N-alkyl acridone derivatives followed the synthetic
procedure described by Wang et. al. [38]. To a mixture of acridone (0.100 g, 0.5 mmol),
K2CO3 (0.0071 g, 0.18 mmol), Al2O3 (0.0533 g, 0.52 mmol) NaOH (0.0071 g, 0.18 mmol)
and TBAB (0.0017 g, 0.005 mmol) the proper alkyl halide, iodomethane, iodopentane or
iodooctane, (2 mL) was added on the 10 mL microwave tube. The mixture was heated
under microwave irradiation during 15 min at 65 ºC with high speed stirring using a CEM
(Discover SP) microwave oven. The reaction mixture was cooled at room temperature and
the reaction product was purified by silica gel column chromatography using ethyl acetate
and n-hexane (3:7) as eluent.
General procedure for the synthesis of N-alkyl-9(10H)-acridone-2,7-disulfonyl
dichloride.
To the N-alkyl-9(10H)acridone (0.100 g) chlorosulfonic acid (2.5 mL, 0.38 mmol)
was added and placed under inert atmosphere in a paraffine bath at 60 °C during 24 h at
vigorous stirring. The solution is left to cool in ice and a saturated solution of NaHCO3 (25
mL) was added. Then 20 mL of chloroform were added and the organic layer extracted.
The organic layer was washed three times with water, dried with sodium sulfate
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anhydrous and filtrated. The evaporation of chloroform under vacuum yield the N-alkyl-
9(10H)-acridone-2,7-disulfonyl dichloride as yellow solids.
General procedure for the synthesisN-alkyl-9(10H)-acridone-2,7-disulfonic acids.
The chlorosulfonic derivatives were hydrolysed in water (50 mL) at 110 °C during
48 h. After cooling to room temperature the solvent was evaporated under vacuum
yielding the corresponding disoulfonic acids.
Characterization
9(10H)-Acridone (from Sigma-Aldrich 99%)
(Yellow solid) Mp. 360-362 °C. 1H NMR (400 MHz, DMSO-d6) δ(ppm) 11.73 (s, 1H), 8.23 (dd, J
= 8.1, 1.3 Hz, 2H), 7.74 (dt, J = 8.4, 1.6 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.26 (dt, J = 8.0, 1.0
Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ(ppm) 176.7, 140.8, 133.4, 125.9, 120.9, 120.4, 117.3.
IR (ATR-FTIR) ν = 2939 (w, broadband), 1630 (m), 1591 (m), 1555 (m), 1528 (s), 1468 (s), 1338
(s), 1257 (m), 1156 (s), 1103 (m), 1021 (m), 934 (s), 812 (m), 747 (s), 669 (s).
9(10H)-Acridone-2,7-disulfonyl dichloride
(Pale Yellow solid)Yield: 28%. Mp. >300°C. H NMR (400 MHz, DMSO-d6) δ (ppm) 11.89 (s,
1H), 8.47 (d, J = 1.6 Hz, 2H), 7.92 (dd, J = 8.6, 1.9 Hz, 2H), 7.49 (d, J = 8.6 Hz, 2H). C NMR
1
13
(101 MHz, DMSO-d6) δ (ppm) 176.9, 141.3, 140.7, 131.2, 122.9, 119.3, 116.9. IR (ATR-FTIR)
ν = 3253 (w), 3218 (w), 3183 (w), 3125 (w), 3020 (w), 1623 (s), 1599 (s) 1584 (m), 1515 (m),
1464 (m), 1375 (s), 1170 (s), 1130 (s), 1071 (s), 988 (m), 908 (m), 823 (s), 741 (m), 698 (s).
MSHR m/z found [M+H]+391.92170 molecular formula C13H8Cl2NO5S2 requires 391.92150.
9(10H)-Acridone-2,7-disulfonic acid
(Yellow solid) Yield: 99%. Mp. >300 °C. H NMR (400 MHz, DMSO-d6) δ(ppm) 11.86 (s, 1H),
8.47 (d, J = 1.1 Hz, 2H), 7.92 (dd, J = 8.6, 1.7 Hz, 2H), 7.49 (d, J = 8.6 Hz, 2H). C NMR (101
1
13
MHz, DMSO-d6) δ(ppm) 176.9, 140.7, 131.2, 123.0, 119.3, 117.0. IR (ATR-FTIR) ν = 3258 (w,
br), 1625 (w), 1585 (w), 1515 (w), 1473 (w), 1089 (s), 1032 (s), 823 (s) 709 (s). MSHR m/z found
[M+H]+355.98871 molecular formula C13H10NO7S2 requires 355.98932.
N-Methyl-9(10H)-Acridone
(Pale Yellow solid) Yield: 71.1%. Mp. 201-204 °C. 1H NMR (400 MHz, CDCl3) δ(ppm) 8.58 (dd,
J = 8.0, 1.6 Hz, 2H), 7.75 (dt, J = 8.7, 1.7 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H), 7.31 (dt, J = 7.9, 0.8
Hz, 2H), 3.92 (s, 3H). 13C NMR (101 MHz, CDCl3) δ(ppm) 178.2, 142.6, 133.8, 127.8, 122.6,
121.3, 114.7, 33.7. IR (ATR-FTIR) ν = 2916 (w), 2847 (w), 1629 (m), 1589 (m), 1486 (m), 1459
(m), 1439 (m), 1366 (m), 1339 (m), 1287 (m), 1178 (m), 1127 (m), 1037 (m), 936 (m), 859 (m),
802 (m), 751 (s), 670 (s). MSHR m/z found [ M+H ]+210.09180 molecular formula C14H12NO
requires 210.09130.
N-Methyl-9(10H)-Acridone-2,7-disulfonyl dichloride
(Pale Yellow solid) Yield: 30%. Mp. > 300 °C (degradation at 200 ºC).1H NMR (400 MHz,
CDCl3) δ(ppm) 9.19 (d, J = 2.5 Hz, 2H), 8.36 (dd, J = 9.2, 2.5 Hz, 2H), 7.80 (d, J = 9.3 Hz, 2H),
4.07 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ(ppm) 176.6, 142.2, 141.4, 131.5, 123.4, 120.5,
115.6, 34.1. IR (ATR-FTIR) ν = 3126 (w), 3075 (w), 2950 (w), 2853 (w), 1650 (m), 1592 (s),
1479 (m), 1371 (s), 1337 (m), 1279 (w), 1171 (s), 1147 (s), 1071 (s), 990 (w), 930 (w), 875 (w),
813 (s), 704 (s). MSHR m/z found [M+H]+405.93560 molecular formula C14H10Cl2NO5S2 requires
405.93720.
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N-Methyl-9(10H)-Acridone-2,7-disulfonic acid
(Yellow solid) Yield: 99%. Mp. > 300 °C (degradation at 200 ºC).1H NMR (400 MHz, D2O)
δ(ppm) 8.65 (d, J = 2.2 Hz, 2H), 8.08 (dd, J = 9.2, 2.3 Hz, 2H), 7.77 (d, J = 9.2 Hz, 2H), 3.87 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ(ppm) 176.6, 142.1, 141.5, 131.5, 123.4, 120.5, 115.8,
34.1. IR (ATR-FTIR) ν = 3062 (w, broadband), 1617 (m), 1586 (m), 1551 (m), 1490 (m), 1282
(m), 1247 (m), 1197 (m), 1144 (m), 1085 (s), 1006 (s), 815 (s), 710 (s), 681 (s). MSHR m/z found
[ M+H]+370.00413 molecular formula C14H12NO7S2 requires 370.00497.
N-Penthyl-9(10H)-Acridone
(Pale Yellow solid) Yield: 66.7%. Mp. 117-119 °C (yellow liquid).1H NMR (400 MHz, CDCl3)
δ(ppm) 8.60 (dd, J = 8.0, 1.7 Hz, 2H), 7.74 (dt, J = 8.7, 1.7 Hz, 2H), 7.51 (d, J = 8.7 Hz, 1H), 7.31
(dt, 11.1, 3.9 Hz, 2H), 4.35 (t, 2H), 2.00-1.92 (m, 2H), 1.60-1.45 (bm, 4H), 1.00 (t, J = 7.1 Hz,
13
3H). C NMR (101 MHz, CDCl3) δ(ppm) 178.0, 141.8, 133.9, 128.1, 122.5, 121.2, 114.5, 46.2,
29.1, 26.9, 22.5, 14.1. IR (ATR-FTIR) ν = 3075 (w), 2953 (w), 2925 (w), 2869 (w), 1631 (m),
1593 (s), 1484 (s), 1452 (s), 1373 (m), 1288 (m), 1262 (s), 1195 (m), 1172 (s), 1045 (m), 971 (m),
937 (m), 847 (m), 764 (s), 747 (s), 671 (s). MSHR m/z found [M+H]+266.15380 molecular
formula C18H20NO requires 266.15390.
N-Penthyl-9(10H)-Acridone-2,7-disulfonyl dichloride
(Pale Yellow solid) Yield: 69.9%. Mp. 188-190 °C.1H NMR (400 MHz, CDCl3) δ(ppm) 9.20 (d, J
= 2.5 Hz, 2H), 8.36 (dd, J = 9.3, 2.5 Hz, 2H), 7.73 (d, J = 9.3 Hz, 2H), 4.44 (t, J = 8.5 Hz 2H),
2.05-1.97 (m, 2H), 1.57-1.48 (m, 4H), 1.02 (t, J = 7.2 Hz, 3H).13C NMR (101 MHz, CDCl3)
δ(ppm) 175.4, 145.3, 138.4, 131.9, 129.0, 122.5, 116.9, 47.7, 28.8, 26.8, 22.4, 13.9. IR (ATR-
FTIR) ν = 3072 (w), 2956 (w), 2928 (w), 2861 (w), 1657 (m), 1592 (m), 1478 (m), 1369 (m), 1276
(w), 1173 (m), 1145 (m), 1075 (m), 820 (m), 688 (m). MSHR m/z found [M+H]+461.99970
molecular formula C18H18Cl2NO5S2 requires 461.99980.
N-Penthyl-9(10H)-Acridone-2,7-disulfonic acid
(Yellow hygroscopic solid) Yield: 69.9%.1H NMR (400 MHz, D2O) δ(ppm) 8.67 (s, 2H), 8.05 (d,
J = 9.0 Hz, 2H), 7.71 (d, J = 9.1 Hz, 2H), 4.27 (t, J = 8.5 Hz 2H), 1.73-1.67 (m, 2H), 1.39-1.24 (m,
4H), 0.80 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ(ppm) 176.5, 141.3, 131.7, 123.6,
120.5, 115.7, 30.6, 28.1, 26.6, 22.0, 13.9. IR (ATR-FTIR) ν = 3250 (w, broadband), 2956 (w),
2928 (w), 2865 (w), 1628 (m), 1584 (m), 1486 (m), 1172 (s), 1096 (s) 1027 (s), 983 (s), 811 (s),
710 (s). MSHR m/z found [M+H]+426.066150 molecular formula C18H20NO7S2 requires
426.067570.
N-Octyl-9(10H)-Acridone
(Pale Yellow solid) Yield: 40%. Mp. 106-108 °C (yellow liquid).1H NMR (400 MHz, CDCl3)
δ(ppm) 8.58 (dd, J = 8.0, 1.7 Hz, 2H), 7.73 (dt, J = 8.7, 1.7 Hz, 2H), 7.49 (d, J = 8.7 Hz, 2H), 7.29
(t, 2H), 4.33 (t, 2H), 1.97-1.89 (m, 2H), 1.61 – 1.50 (m, 2H), 1.50 – 1.40 (m, 2H), 1.38-1.29 (m,
4H), 0.91 (t, J = 6.9 Hz, 3H).13C NMR (101 MHz, CDCl3) δ (ppm) 178.0, 141.8, 133.9, 128.0,
122.5, 121.2, 114.5, 46.2, 31.8, 29.3 (d), 27.2, 26.9, 22.6, 14.1. IR (ATR-FTIR) ν = 2955 (w),
2912 (w), 2851 (w), 1630 (m), 1596 (m), 1493 (m), 1462 (m), 1378 (m), 1290 (m), 1266 (m), 1180
(m), 1044 (m), 938 (m), 747 (s), 669 (s). MSHR m/z found [M+H]+308.20070 molecular formula
C21H26NO requires 308.20090.
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Voltammetric parameters and electrochemical cells
The 0.1 M ionic strength electrolyte solutions [39] at pH 0.0 H2SO4, pH 6.9
phosphate buffer and pH 13.0 NaOH/KCl were prepared using analytical grade reagents
and purified water from a Millipore Milli-Q system (conductivity ≤ 10 µS/cm). Solutions
of different concentrations were obtained by dilution of the appropriate volume in the
supporting electrolyte. Nitrogen-saturated solutions were obtained by bubbling high purity
N2 for a minimum of 10 min in the solution and continuous flow of pure gas over the
solution during the voltammetric experiments. All experiments were done at room
temperature, T=298 K (25 °C). Microvolumes were measured using EP-10 and EP-100
Plus Motorized Microliter Pipettes (Rainin Instrument Co. Inc., Woburn, USA). The pH
measurements were carried out with a Crison micropH 2001 pH-meter with an Ingold
combined glass electrode. Voltammetric experiments were carried out using an Autolab
potentiostat with GPES 4.9 software (Eco-Chemie, Utrecht, The Netherlands). The
measurements were carried out in a solution volume of 3 mL, using a three-electrode
system one-compartment electrochemical cell of capacity 2 mL.
A glassy carbon electrode (GCE, d = 1.5 mm) was the working electrode, a Pt wire
the counter electrode and an Ag/AgCl (3 M KCl) reference electrode (Bio-Logic SAS,
France). The experimental conditions were as follows: for cyclic voltammetry (CV), scan
rate 100 mV/s; for differential pulse (DP), voltammetry pulse amplitude 50 mV, pulse
width 70 ms and scan rate 5 mV/s. The glassy carbon electrode (GCE) was polished using
a diamond spray (particle size 3 µm) (Kemet, UK) before each electrochemical
experiment. After polishing, it was rinsed thoroughly with Milli-Q water. Following this
mechanical treatment, the GCE was placed in a buffer supporting electrolyte, and
differential pulse voltammograms were recorded until a steady-state baseline
voltammogram was obtained. This procedure ensured very reproducible experimental
results. Differential pulse voltammograms presented were background subtracted and
baseline corrected using the moving average application with a step window of 5 mV
included in the GPES v4.9 software.
This mathematical treatment improves the visualization and identification of peaks
over the baseline without introducing any artefact, although the peak intensity is, in some
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cases, reduced (10%) relative to that of the untreated curve. Nevertheless, this
mathematical treatment of the original voltammograms was used in the presentation of all
experimental voltammograms for an improved and clearer identification of the peaks. The
values for peak current presented in all plots were determined from the original untreated
voltammograms after subtraction of the baseline.
RESULTS AND DISCUSSION
Synthesis
N-alkyl-9(10H)-acridones were obtained adapting the method described by Wang et
al.[38] for the synthesis of these compounds using a domestic microwave oven. The alkyl
derivatives were obtained from a mixture of AC, Al2O3, NaOH, a catalytic amount of
tetrabutylammonium (TBAB) and the respective alkyl halide: CH3I, nC5H11I or nC8H17I in
excess to act as solvent and reactant. Using a microwave reactor for synthesis with
continuous monitoring of power, temperature and pressure it was possible obtain the
desired products after 15 min at 65 ºC with initial power of 100 W, avoiding the use of
solvent and in short reaction times when compared with conventional methodologies[40-
42]. After purification by column chromatography the N-alkyl derivatives were obtained
in moderate to high yields.
The chlorosulfonic derivatives of N-alkyl-9(10H)-acridones were obtained by
reaction in neat chlorosulfonic acid at 60 ºC for 24 hours under vigorous stirring in N2
atmosphere [33]. The yellow solution was then neutralized with a saturated solution of
NaHCO3 and the desired product extracted with chloroform obtaining the chlorosulfonic
derivatives with yields ranging 28.0-69.9%. It was not possible to obtain the
chlorosulfonic derivative of N-octyl-9(10H)-acridone by chlorosulfonation using different
reaction conditions, in all cases a black solid insoluble in organic solvents and water was
obtained.
The hydrolyses of the sulfonyl chloride acridones performed by refluxing in water
during 48 h yield the water-soluble sulfonic acids in quantitative yields, Scheme 2.
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Scheme 2: Schematic reaction pathways and reaction yield for the synthesis of the N-
alkylacridones, sulfonyl chloride acridone derivatives and the water-soluble acridone sulfonic acid
derivatives.
Spectral and Photophysical data
Figures 1 and 2 depict respectively the absorption and fluorescence spectra of the
three families of compounds, the (i) N-alkylacridones, the (ii) chlorosulfonic and (iii)
sulfonic acid derivatives. The electronic spectral properties are summarized in Table 1.
The introduction of the alkyl chain in AC, ACSO2Cl and ACSO3H lead to the red
shift of the absorption wavelength maxima in DMSO of ca. 5 nm, regardless the length of
the chain, and to an increase of the molar extinction coefficient. In water, the same effect
can be observed for AC and ACSO3H derivatives with an increase of the red shift from 7
to 9 nm. Introduction of the chlorosulfonic and sulfonic acid substituents does not affect
the absorption wavelength maxima but has a significant effect in the molar extinction
coefficient decreasing from 9900 mol-1dm3cm-1 for AC to 6000 mol-1 dm3 cm-1 for
ACSO3H.
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1.0
0.8
0.6
0.4
0.2
0.0
A
B
C
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
275 300 325 350 375 400 425 450
λ(nm)
Figure 1: Absorption spectra of Acridone and its chlorosulfonic and sulfonic acid derivatives in
DMSO at T=294K; A) black line: AC, red line: MetAC, blue line: PetAC, magenta line: OctAC.
B) black line: ACSO2Cl, red line: MetACSO2Cl, blue line: PetACSO2Cl. C) black line: ACSO3H,
red line: MetACSO3H, blue line: PetACSO3H.
The fluorescence emission maximum in DMSO displays a red-shift with the
introduction of the alkyl chain, higher for N-methylacridone leading to also an increase in
the Stoke’s Shift value. This effect is further intensified on going from the less polar
DMSO (solvent’s permittivity ε’=47.24) to the more polar solvent water (ε’=78.54)
achieving Stoke’s shift values of ~ 2000 cm-1. A less pronounced effect is observed with
the chlorosulfonic acid derivatives, Table 1.
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1.0
0.8
0.6
0.4
0.2
0.0
A
B
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
C
400 425 450 475 500 525 550
λ(nm)
Figure 2: Emission spectra of AC and its derivatives in DMSO at T= 294 K. A) black line: AC,
red line: MetAC, blue line: PetAC, magenta line: OctAC. B) black line: ACSO2Cl, red line:
MetACSO2Cl, blue line: PetACSO2Cl. C) black line: ACSO3H, red line: MetACSO3H, blue line:
PetACSO3H.
Table 1: Spectroscopic data including absorption (λabs(nm)) and fluorescence emission (λfluo(nm))
wavelength maxima, Stokes Shift (∆SS) and molar extinction coefficients (ε) obtained for the
investigated Acridones and its chlorosulfonic and sulfonic acid derivatives in DMSO and Water
(pH=5.5) at T=293 K. The dielectric constant of the solvents (ɛ ') is also presented.
Stokes
Compound Solvent ɛ '(25º C)
λabs(nm)
λfluo(nm)
ɛ(L.mol-1.cm-1)
∆
SS
Shift
293 K
293 K
(nm)
(cm-1)
DMSO
H2O
47.24
80.4
398
385
408
416
10
31
616
1936
9900
-
AC
DMSO
H2O
47.24
80.4
403
394
417
430
14
36
833
2125
10490
-
MetAC
11

ACCEPTED MANUSCRIPT
DMSO
H2O
47.24
80.4
403
394
414
428
11
34
659
2016
11290
-
PetAC
OctAC
DMSO
H2O
47.24
80.4
404
ND
414
ND
10
ND
598
ND
11680
ND
DMSO
DMSO
DMSO
47.24
47.24
47.24
399
406
406
417
424
422
18
18
16
1082
1046
934
7240
8690
8420
ACSO2Cl
MetACSO2Cl
PetACSO2Cl
DMSO
H2O
47.24
80.4
399
396
417
417
18
21
1082
1272
6000
4430
ACSO3H
MetACSO3H
PetACSO3H
DMSO
H2O
47.24
80.4
405
403
424
433
19
30
1106
1719
7490
5610
DMSO
H2O
47.24
80.4
406
406
421
431
15
25
878
1429
7400
6190
ND= not determined due to the poor solubility of this compound in water.
Photophysical Properties
The quantum yield (φF) and lifetime (τF) of fluorescence together with the radiative
(kF) and radiationless (kNR) rate constants at T= 293 K, obtained for the investigated
acridones (AC, MetAC, PentAC, and OctAC) and its chlorosulfonic (ACSO2Cl,
MetACSO2Cl and PetACSO2Cl) and sulfonic acid (ACSO3H, MetACSO3H and
PetACSO3H) derivatives in DMSO and water (pH=5.5, only for the water soluble
derivatives) are summarized in Table 2.
The introduction of the alkyl group in the acridone ring influences the fluorescence
quantum yield, which decreases from 0.75 (AC) to 0.65, 0.68 and 0.66 for the methyl
penthyl and octyl substituted acridone respectively. The fluorescence lifetime also shows a
12

ACCEPTED MANUSCRIPT
gradual decrease with the increase of the alkyl chain. The clorosulfonic and sulfonic acid
derivatives display a longer lifetime in DMSO increasing the fluorescence quantum yield
up to 0.70 (PetACSO3H).
Figure 3- Picture of the studied compounds obtained under day light exposure (top picture)
and (bottom picture) under UV light (365 nm). On the left ACSO2Cl, MetACSO2Cl and
PetACSO2Cl in DMSO. In the middle ACSO3H, MetACS3H, PetACSO3H in DMSO and on right
ACSO3H, MetACS3H and PetACSO3H in water (pH=5.5).
Solubilization in water increases the fluorescence quantum yield up to 0.8 (0.82 for
ACSO3H and 0.92 for PetACSO3H) more than 10% comparing with the fluorescence in
DMSO, and also increases the decay time values; these are longer than 15 ns (16.49 ns for
ACSO3H and 15.62 ns for PetACSO3H). It is also worth noting the constancy of the
radiative (kF) and radiationless (kNR) rate constants for all the compounds and solvents.
This shows that although the water-soluble acridones (ACSO3H, MetACSO3H and
PetACSO3H) display longer lifetimes the balance between the radiative and radiationless
channels is kept constant and so is the dominant deactivation mechanism. Moreover, there
is also a significant difference between the value in DMSO vs. water, showing that the
compounds are sensitive to the media (polarity and pH) and adequate to use this
compounds as lifetime fluorescence probes, Table 2. Although the mechanism behind this
effect has not been investigated in detail in this work, it is likely that it involves the close
proximity between two excited singlet excited states of S1 (π,π*) and S2 (π,π* with a
contribution of an n,p* state) in which the S2 becomes destabilized in water (relative to
DMSO) and the S1 becomes more allowed in nature.[5]
13

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The different decay time of the water-soluble acridones (ACSO3H, MetACSO3H
and PetACSO3H) in water vs. DMSO lead us to further explore the pH dependence
behaviour of these compounds.
Table 2: Photophysical parameters including the fluorescence quantum yield (φF) and
fluorescence lifetime (τF) together with the radiative (kF)and radiation less (kNR) rate constants
obtained for the investigated Acridones and its chlorosulfonic and sulfonic acid derivatives in
DMSO and Water (pH=5.5) at T=293 K.
*
*
kF(ns-1) kNR (ns-1)
Compound
Solvent
φF
τF (ns)
293 K
293K
DMSO
DMSO
DMSO
DMSO
0.75
0.65
0.68
0.66
9.10
8.85
8.10
7.96
0.082
0.073
0.084
0.083
0.027
0.040
0.040
0.043
AC
MetAC
PetAC
OctAC
Compound
Solvent
τF (ns)
kF(ns-1) kNR (ns-1)
φF
293 K
293K
DMSO
DMSO
DMSO
0.70
0.70
0.76
10.78
10.59
10.08
0.065
0.066
0.075
0.028
0.028
0.024
ACSO2Cl
MetACSO2Cl
PetACSO2Cl
Compound
Solvent
τF (ns)
kF(ns-1) kNR (ns-1)
φF
293 K
293K
DMSO
H2O
0.66
0.82
10.95
16.49
0.060
0.050
0.031
0.011
ACSO3H
DMSO
H2O
0.64
0.76
10.64
16.71
0.060
0.045
0.034
0.014
MetACSO3H
PetACSO3H
DMSO
H2O
0.70
0.92
10.17
15.62
0.069
0.059
0.029
0.005
φF
τF
1−φF
*
;
kF =
kNR =
τF
14

ACCEPTED MANUSCRIPT
The spectra and data are summarized in Figure 4 and Table 5 respectively. Figure 4
shows a clear shift of the wavelength maxima and shape upon going from pH=0 (and 6.9)
to alkaline pH (~13) values. Indeed, whereas from pH=0 to 6.9 the absorption spectra is
basically identical at the two pH values, there is a small increase of the vibronic resolution
at pH=6.9. In addition the energy of the first singlet excited state, S1, (E0-0) obtained from
the overlap of the normalized absorption and emission spectra is identical at these two pH
values (E0-0=107 (408, 409 nm) = 24450 cm-1, 24510 cm-1) whereas at alkaline pH values
(pH=13) the absorption and emission spectra together with the energy of S1 state, E0-0
(24969 cm-1), clearly indicates the presence of a new species.
1.2
0.8
0.4
0.0
1.2
0.8
0.4
0.0
Abs
Em 376
Exc 490
A
300
350
400
450
500
550
600
650
1.2
0.8
0.4
0.0
1.2
0.8
0.4
0.0
Abs
B
Em 376
Exc 490
300
350
400
450
500
550
600
650
1.6
1.2
0.8
0.4
0.0
1.6
1.2
0.8
0.4
0.0
Abs
Em 376
Exc 520
C
300
350
400
450
500
550
600
650
λ(nm)
Figure 4: Absorption and emission spectra of the sulfonated Acridone ACSO3H in water at
T=293 K and A) pH=0; B) pH=6.9 and C) pH=13.
However, the most interesting feature comes from the analysis of the photophysical
parameters in Table 3. Indeed, as can be seen from Table 3, although the φF value is
15

ACCEPTED MANUSCRIPT
constant with pH (exception made for the higher pH value, where the φF decreases), the
same does not occur with the τF value thus showing that this compound can be potentially
used as a lifetime probe.
Table 3–Photophysical parameters including the fluorescence quantum yield (φF) and
fluorescence lifetime (τF) together with the radiative (kF) and radiationless (kNR) rate
constants obtained for ACSO3H at different pH values.
φF
τF (ns)
kF(ns-1)
kNR (ns-1)
pH
0.0
5.5
6.9
13
0.82
0.81
0.81
0.73
18.74
16.28
17.24
22.3
0.0438
0.0498
0.0470
0.0327
0.0096
0.0117
0.0110
0.0121
φF
τF
1−φF
kF =
;
kNR =
τF
Electrochemical behavior
The influence of the substituents in the acridone moitey, namely in its redox
properties, was further investigated, using cyclic and differential pulse voltammetry at a
glassy carbon electrode. The electrochemical behavior of the water soluble sulfonated
acridones was investigated in water at different pH values. Only the 9(10H)-Acridone-2,7-
disulfonic acid (ACSO3H) presented oxidation and reduction potentials in the range -1.25
V to + 1.25 V. A 0.3 mM stock solution of the ACSO3H was freshly prepared prior to use.
The data obtained are summarized in Table 4.
Table 4 – Electrochemical data including the oxidation (Epa) and reduction (Epc) potentials
for the three sulfonated acridones at three pH values. As mentioned for MetACSO3H and
PetACSO3H no oxidation or potential could be observed in the -1.25 V to +1.25 V range.
See text for more details.
16

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Compound
Ep
pH = 0.0
pH = 6.9
Epa1 =1.177V
ACSO3H
Epa1 = 1.064 V
Epc2 = - 0.641 V
pH = 12.8
Epa1 = 0.808 V
-
-
MetACSO3H
PetACSO3H
1
Epa = oxidation potential for the first peak in the voltammogram in Fig. 4
2
Epc =reduction potential for the second peak in the voltammogram in Fig. 4
The voltammetric behavior of ACSO3H, MetACSO3H or PetACSO3H at a
concentration of 437 µM was investigated using a GCE in a solution 0.1 M phosphate
buffer solution (saturated with N2) at pH 6.9. The cyclic voltammograms obtained for
1
ACSO3H at a scan rate ʋ = 100 mV s-1, shows a single peak in the anodic region (Epa ) at
1.064 V (Figure 4); no peaks were observed in the cathodic region, indicating that the
oxidation processes are irreversible. Neither MetACSO3H nor PetACSO3H showed redox
activity thus indicating the involvement of the NH group in the redox process.
1
a
2
c
5 µΑ
-1.0
-0.5
0.0
0.5
1.0
E / V (vs. Ag/AgCl)
Figure 4 - Cyclic voltammograms of a solution of ACSO3H(0.3 mM ) at pH 7 (0.1 M
phosphate buffer): A) (—) first and ( ····· ) second scan, ʋ =100 mV s-1.
On running the second CV, obtained from the ACSO3H solution at pH= 6.9, without
2
cleaning the GCE surface, a new reduction peak (indicated as 2c in Fig. 4), at Epc = -
0.641 V appeared. This peak is related to the irreversible reaction of the ACSO3H
oxidation product formed at the GCE surface during the first scan. The decrease of the
17

ACCEPTED MANUSCRIPT
current of peak 1a observed when the second scan finishes is explained by the adsorption
of ACSO3H and of its oxidation products at the electrode surface.
The oxidation of ACSO3H is linearly pH-dependent (variation of peak 1a potential
with pH), giving rise to the equation Epa (V) = 0.931 – 0.059 pH. The slope of the line, 59
mV per pH unit, indicates that the same number of proton and electron are involved in the
oxidation process of ACSO3H. [43]
Furthermore, the number of electrons transferred, n, was determined from the peak
width at half height, W1/2 = 91 mV, and is close to the theoretical value of 90 mV,
corresponding to an electrochemical reaction involving the transfer of a single electron
[43]. Consequently, it can be concluded that the oxidation process for ACSO3H above
described involves the simultaneous transfer of one electron and one proton.
The energies of the HOMO and LUMO levels were obtained for ACSO3H at
different pH values from equations 1 and 2. The energy of the HOMO, EHOMO, was
calculated from the onset oxidation potential, Eonset(ox), plus 4.8, which is the reference
energy level of ferrocene below the vacuum level and the oxidation potential of ferrocene,
EFOC, given by the potential of FOC/FOC+vs Ag/AgCl measured by cyclic voltammetry at
each pH value. The LUMO energy, ELUMO, was obtained from the optical energy gap E0-0,
measured from the interception of the normalized UV-Vis absorption and fluorescence
spectra, through equation 2 at each pH value. The data are summarized in Table 5.
onset
EHOMO = −(E
+ 4.8)− EFOC (1)
ox
ELUMO = EHOMO + E0−0
(2)
Table 5 –Experimental parameters needed to obtain the HOMO and LUMO energy values
for ACSO3H at different pH values obtained from eq. (1) and (2) respectively.
E0-0 (eV)b ELUMO
(eV)
λ0-0 (nm)a
pH
Eonset(ox)
EFOC(V)
EHOMO
(eV)c
(V)
0.0
1.100
1.000
0.696
0.395
0.224
0.402
-5.505
-5.576
-5.094
409
408
400
3.031
3.039
3.099
-2.474
-2.537
-1.995
6.9
12.8
aWavelength of the 0-0 electronic transition resulting from the intersection of the
normalized absorption and the emission spectra.
18

ACCEPTED MANUSCRIPT
bE0-0 values resulting from the intersection of the normalized absorption and the emission
spectra.
Although we have developed a way to make acridones water soluble, the fact that
the compounds show irreversible waves is an obvious limitation for Redox Flow Batteries
(RFB) applications. Further substitution on the sulfonated acridones may induced the
desired reversibility for RFB. However, the study of ACSO3H at different pH indicates
that the LUMO energy levels are, in absolute value, higher than the TiO2 conduction-band
(CB) value (-4.26 eV[44], with values ranging from −4.0 to −4.3 eV[45, 46]) and could
therefore make efficient organic dye photosensitizers for dye sensitized solar cells
(DSSCs) since they have enough driving force for an effective injection into the CB of
TiO2.
CONCLUSIONS
A comprehensive investigation of the electronic spectral, photophysical and
electrochemical properties of three different families of acridone derivatives has been
undertaken in organic solvents and aqueous solutions at different pH values. Although
stables when submitted to various electrochemical cycles, due to the lack of irreversible
waves the compounds show limitation for RFB uses. Nonetheless, the present work shows
that these compounds are highly fluorescent and have their photophysical parameters
dependent on the polarity and pH values of the media. In particular with the water-soluble
ACSO3H it is shown that its long fluorescent decay time value, which is shown dependent
on the pH, can constitute a lifetime probe both for in vitro and in vivo studies.
ACKNOWLEDGEMENTS
We acknowledge funding by FEDER (Fundo Europeu de Desenvolvimento
Regional) through COMPETE (Programa Operacional Factores de Competitividade). The
Coimbra Chemistry Centre is supported by the Fundaçãoo para a Ciência e a Tecnologia
(FCT), Portuguese Agency for Scientific Research, through the program
UID/QUI/UI0313/2013. This work was performed under the project “SunStorage -
19

ACCEPTED MANUSCRIPT
Harvesting and storage of solar energy”, with reference POCI-01-0145-FEDER-016387,
funded by European Regional Development Fund (ERDF), through COMPETE 2020 -
Operational Programme for Competitiveness and Internationalization (OPCI), and by
national funds, through FCT - Fundação para a Ciência e a Tecnologia I.P. RCP and
ADRP acknowledge Sunstorage for research (BI) and pos-doc grants respectively.
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Highlights
New water-soluble acridones have been synthesized
The displayed photophysical properties have shown these as potential polarity
and pH probes
The electrochemical properties have shown these as potential dyes for DSSC