Novel fluorescent probes for L-cysteine based on the xanthone skeleton

Article abstract of DOI:10.1016/j.jphotochem.2019.112153

Two novel derivatives of xanthone (9H-xanthen-9-one, dibenzo-γ-pyrone) containing a maleimide moiety have been synthesized. Their properties were characterized by the combination of NMR, MS, electronic absorption and fluorescence spectroscopy. The reactiv

Full text of DOI:10.1016/j.jphotochem.2019.112153

JournalofPhotochemistry&PhotobiologyA:Chemistry387(2020)112153
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Journal of Photochemistry & Photobiology A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Novel fluorescent probes for L-cysteine based on the xanthone skeleton
Aleksandra Grzelakowskaa,⁎, Jolanta Kolińskaa,⁎, Małgorzata Zakłos-Szydab, Jolanta Sokołowskaa
a Institute of Polymer and Dye Technology, Faculty of Chemistry, Lodz University of Technology, Stefanowskiego 12/16, Lodz, 90-924, Poland
b Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, Lodz, 90-924, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Fluorescent probes
Turn on
L-cysteine
Thiols
Two novel derivatives of xanthone (9H-xanthen-9-one, dibenzo-γ-pyrone) containing a maleimide moiety have
been synthesized. Their properties were characterized by the combination of NMR, MS, electronic absorption
and fluorescence spectroscopy. The reactivity of these compounds toward L-Cys as well other analytes was de-
termined. The results show that the novel derivatives of xanthone demonstrate a high “turn-on” fluorescence
response and selectivity toward L-cysteine and have the potential to act as probes to L-cysteine under physio-
logical conditions. Reaction of 2-maleimidoxanthone (4a) and 2,7-dimaleimidoxanthone (4b) with L-Cys lead to
the formation of the fluorescent products. In the presence of L-Cys, the fluorescence intensities of probes 4a and
4b have greatly enhanced 25-fold and 60-fold, respectively. Finally, the probes 4a-4b were used to detection of
thiols in the human cell line HeLa.
Xanthone
1. Introduction
reactions involving their double bond. In most cases, they do not ex-
hibit fluorescence, but the strong fluorescence is characterized of their
Thiols are important molecules in intracellular milieu and in bio-
logical processes. Cysteine (Cys), homocysteine (Hcy) and glutathione
(GSH) significantly affect many cellular functions. For example, GSH is
one of the major endogenous antioxidants and is capable of preventing
cellular component damage caused by reactive oxygen species. The
ratio of GSH (reduced) and its disulfide, GSSG (oxidized), contributes to
the redox potential of the cell and redox homeostasis [1,2]. Cysteine
plays crucial role in the protein synthesis, detoxification, and metabolic
processes [3]. Moreover, the altered levels of the thiols have been
linked with several human diseases and consequences of diseases [4].
For these reasons, the detection of thiol biomolecules is vital. Fluor-
escent probes have emerged as a versatile tool for the detection of
thiols. Because of the high sensitivity of fluorescent methods, fluoro-
genic probes for thiols became important tools in the studies on these
analytes. The number of fluorogenic probes designed and synthesized
for the detection of thiols is relatively large [5–13]. The mechanisms of
action of these probes are based on various thiols-triggered reactions
including nucleophilic addition [14], nucleophilic substitution [15,16],
cyclisation with an aldehyde [17,18], a cleavage reaction by thiols
[19,20], and other reactions [21–25]. An ideal fluorescent probe should
possess some outstanding properties, such as high sensitivity and se-
lectivity, short response time and proper optical properties [26].
N-substituted maleimides have been widely used in biological fields
as thiol reagents. They react quite selectively with thiols via addition
thiol adduct products [27–30].
In this study, the xanthone was selected as the fluorophore due to its
desirable photophysical properties, such as fluorescence with the
maximum located in the visible region of the spectrum, high fluores-
cence quantum yield and large Stokes shift. The xanthones are a class of
oxygen containing compounds with a broad range of biological activ-
ities such as anti-malarial, anti-tumor [31], anti-cancer [32], anti-bac-
terial [33], anti-oxidant [34,35] and anti-inflammatory [36]. Xan-
thones are also considered as inhibitors of different enzymes [37–45].
The main objectives of 9H-xanthen-9-ones synthesis are not only the
development of more complex and varied bioactive compounds and
structure-activity relationship studies [46]. Because of their excellent
photochemical properties, derivatives of xanthone are used as fluor-
escent dyes and probes [47–49]. Some of them can be applied in
electroluminescent devices [50] and as optical probes for metal ions
[51], reactive oxygen species [52] or the RNA and DNA structure
[53,54]. They are widely seen in a number of natural products [55,56].
Synthetic and naturally occurring xanthone derivatives exhibit diverse
biological activities, which depend on the various substituents and their
positions. The modifications of natural xanthone derivatives are aimed
at expanding the range of their application [57,58].
The current study is a continuation of the authors’ research into
probes and chemosensors for the detection of biothiols [17,59–62]. In
the present investigation, novel derivatives of xanthone were
⁎ Corresponding authors.
E-mail addresses: aleksandra.grzelakowska@p.lodz.pl (A. Grzelakowska), jolanta.kolinska@p.lodz.pl (J. Kolińska).
https://doi.org/10.1016/j.jphotochem.2019.112153
Received 26 June 2019; Received in revised form 14 September 2019; Accepted 8 October 2019
Availableonline13October2019
1010-6030/©2019ElsevierB.V.Allrightsreserved.

A. Grzelakowska, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry387(2020)112153
liquid chromatography (HPLC). Column chromatography purifications
were performed on Silica gel 60 (0.063-0.200 mm) from Merck. TLC
was carried out on Merck TLC Silica gel 60 F254 aluminum sheets. The
spots were visualized by illumination with a UV lamp (λ = 254/
365 nm). HPLC analyses were performed on a Shimadzu instrument
equipped with a diode array detector (DAD, SPD-M20A). The com-
pounds were loaded onto Kinetex C18 column (Phenomenex,
100 mm × 4.6 mm, 2.6 μm) equilibrated with 10% acetonitrile in
water, containing 0.1% trifluoroacetic acid. The products were eluted
by an increase of the acetonitrile concentration from 10 to 100% over
10 min. The flow was kept at 1.5 mL/min. The probes were detected by
monitoring absorbance at 350 nm.
The synthesized compounds were identified and characterized by
proton nuclear magnetic resonance spectra (1H NMR), atmospheric
pressure chemical ionisation mass spectra (APCI MS), and by their
melting point. NMR spectra were recorded with a Bruker Avance DPX
250 spectrometer. Solutions were prepared in CDCl3, acetone-d6 or
DMSO-d6 as a solvent, using tetramethylsilane (TMS) as internal stan-
dard. Chemical shifts (δ) are reported in parts per million (ppm), and
coupling constant (J) values in hertz (Hz). NMR peak multiplicities are
described as follows: s – singlet, d – doublet, dd – doublet of doublets
and m – multiplets. High-resolution mass spectra were obtained with a
Finnigan MAT 95 (Thermo Fisher Scientific, USA) apparatus equipped
with an atmospheric-pressure chemical ionisation method. Melting
points were determined on Boeöthius melting point apparatus – PGH
Rundfunk, Fernsehen Niederdorf KR, Sollberg/E and uncorrected.
2a: yield: 1.40 g (29%), m.p. 199–200 °C (200–204 °C [63]), Rf 0.75
[toluene/ethanol 3:1 (v/v)].
2b: yield: 1.29 g (58%), m.p. 264–265 °C (262–265 °C [63]), Rf 0.69
[toluene/ethanol 3:1 (v/v)].
3a: yield: 0.28 g (46%), m.p. 209–211 °C (210–212 °C [64]), Rf 0.56
[toluene/ethanol 3:1 (v/v)]. 1H NMR (250 MHz, CDCl3): δ 8.26 (dd, J1
= 8.0 Hz, J2 = 1.7 Hz, 1 H), 7.67-7.59 (m, 1 H), 7.48 (d, J = 2.8 Hz,
1 H), 7.40 (d, J = 8.4 Hz, 1 H), 7.33-7.22 (m, 2 H), 7.05 (dd, J1
8.9 Hz, J2 = 2.9 Hz, 1 H), 3.78 (bs, 2 H).
=
3b: yield: 0.34 g (35%), m.p. 272–274 °C (269–271 °C [54]), Rf 0.47
[toluene/ethanol 3:1 (v/v)]. 1H NMR (250 MHz, DMSO-d6): δ 7.22 (d, J
= 8.9 Hz, 2 H), 7.12 (d, J = 2.8 Hz, 2 H), 6.98 (dd, J1 = 8.9 Hz, J2
2.9 Hz, 2 H), 5.23 (s, 4 H).
=
Scheme 1. Synthetic pathway used to obtain novel fluorescent probes based on
a xanthone skeleton (4a-4b).
2.2.1. Synthesis of 2-maleimidoxanthone (4a)
synthesized. The intention was to evaluate the spectroscopic properties
and the study on the reactivity of novel xanthone-based probes toward
L-Cys and other amino acids. These probes contain a maleimide moiety
in the structures that should act as a Michael acceptor. The structures
and route for the synthesis of the studied compounds: 2-maleimidox-
anthone (4a) and 2,7-dimaleimidoxanthone (4b), are shown in Scheme
1.
2-aminoxanthone 3a (0.21 g, 1 mmol) and maleic anhydride
(0.15 g, 1.5 mmol) were refluxed in chloroform (15 mL) until the dis-
appearance of 3a. The progress of the reaction was monitored by TLC
[Merck silica gel 60, solvent acetonitrile/dichloromethane 1:1 (v/v)].
After 2 h, the resulting pale precipitate was filtered, washed with
chloroform (2 × 10 mL), and dried. Acetic anhydride (10 mL) and so-
dium acetate (0.041 g, 0.5 mmol) were added to the obtained solid
(0.28 g), and the mixture was further heated at 100 °C for 4 h. After this
time the reaction mixture was concentrated under vacuum and the
precipitate was filtered, washed with water (25 mL), and dried on the
air. The crude product was purified by column chromatography [acet-
onitrile/dichloromethane 1:1 (v/v)] and crystallization from acetone.
Yield: 0.15 g (52%), m.p. 175 °C, Rf 0.63 [toluene/ethanol 3:1 (v/v)].
HPLC: 99.7% purity, tr = 5.55 min. 1H NMR (250 MHz, acetone-d6): δ
8.24 (dd, J1 = 6.6 Hz, J2 = 2.1 Hz, 2 H), 7.91-7.81 (m, 2 H), 7.72 (d, J
= 9.0 Hz, 1 H), 7.63 (d, J = 8.5 Hz, 1 H), 7.52-7.42 (m, 1 H), 7.08 (s,
2. Experimental
2.1. Materials
Unless otherwise noted, all chemicals were used as received from
commercial sources without further purification (Sigma-Aldrich,
Poland).
2 H). HR-APCI-MS: calculated for ([M+H)+
292.0622.
] 292.0610, found
2.2. Synthesis and characterization of probes
2-nitroxanthone (2a), 2,7-dinitroxanthone (2b), 2-aminoxanthone
(3a) and 2,7-diaminoxanthone (3b) were synthesized according to the
procedures described in the literature with minor modifications [54].
Synthesized compounds 3a-3b and 4a-4b were purified by column
chromatography. The purity of probes 4a-4b was verified using thin-
layer chromatography (TLC) and was checked by high-performance
2.2.2. Synthesis of 2,7-dimaleimidoxanthone (4b)
2,7-diaminoxanthone 3b (0.23 g, 1 mmol) and maleic anhydride
(0.30 g, 3 mmol) were refluxed in chloroform (20 mL) until the dis-
appearance of 3b. The progress of the reaction was monitored by TLC
[Merck silica gel 60, solvent acetonitrile/dichloromethane 1:1 (v/v)].
After 5 h, the resulting pale precipitate was filtered, washed with
2

A. Grzelakowska, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry387(2020)112153
chloroform (2 × 10 mL), and dried. Acetic anhydride (15 mL) and so-
dium acetate (0.062 g, 0.75 mmol) were added to the obtained solid
(0.36 g), and the mixture was further heated at 100 °C for 6 h. After this
time the reaction mixture was concentrated under vacuum and the
precipitate was filtered, washed with water (25 mL), and dried on the
air. The crude product was purified by column chromatography [acet-
onitrile/dichloromethane 1:1 (v/v)] and crystallization from acetone.
Yield: 0.28 g (72%), m.p. 211 °C, Rf 0.58 [toluene/ethanol 3:1 (v/v)].
HPLC: 99.8% purity, tr = 5.18 min. 1H NMR (250 MHz, DMSO-d6): δ
8.69 (d, J = 2.5 Hz, 2 H), 8.35 (dd, J1 = 9.0 Hz, J2 = 2.5 Hz, 2 H), 8.25
(d, J = 9.0 Hz, 2 H), 7.61 (s, 4 H). HR-APCI-MS: calculated for [(M
+H)+] 387.0617, found 387.0619.
treated with 0–25 μM compounds 4a-4b and were incubated for 20 min.
at 37 °C. Subsequently, the fluorescence intensity at 460/40 nm was
measured (λex = 360/40 nm).
The results are expressed as optical density ratio of the treatment to
control.
All data are presented as means
SD.
Microscopic images were taken using fluorescent microscope Nikon
TS100 Eclipse (Tokyo, Japan) at 100× magnification with excitation
filter at 340–380 nm.
3. Results and discussion
3.1. Synthesis and spectroscopic characterization of novel probes
2.3. Spectroscopic measurements
The synthetic route of probes 4 is illustrated in Scheme 1. Firstly,
compounds 2a-2b were achieved by nitration of xanthone [54]. Then,
compounds 3a-3b were synthesized by reduction of the nitro groups to
amino moieties [54]. Finally, probes 4a-4b were obtained by a two
stage synthesis. The amines 3a-3b were reacted with maleic anhydride
in chloroform and subsequently with sodium acetate and acetic anhy-
dride yielding 4a-4b. The progress of the reaction was controlled by
TLC chromatography. The crude probes 4a-4b were purified by column
chromatography. The purity was checked by HPLC. All compounds
were synthesized in acceptable yields (52–72%). The chemical struc-
tures were confirmed with 1H NMR spectroscopy and APCI mass
spectrometry. The chemical shifts, multiplicities and integration of the
relevant groups of protons are in accordance with the structure of the
molecules. The corresponding 1H NMR spectra and APCI mass spectra
are shown in the Supplementary Information (Figs. S1–S4). It is worth
emphasizing that the presented method of xanthone derivatives
synthesis is simple and the obtained compounds can be easily purified
using standard laboratory techniques.
Jasco V–670 UV–vis/NIR spectrophotometer (Jasco, Japan) was
used to record steady-state absorption spectra and FLS-920 spectro-
fluorimeter (Edinburgh Instruments, UK) to measure steady-state
emission spectra and fluorescence lifetimes [65,66]. Spectroscopic
measurements were performed in a standard rectangular quartz cell (10
mm × 10 mm, 3.5 mL). To record an emission spectrum, the excitation
wavelength of the absorption maximum was used (emission/excitation
slit = 1nm).
Fluorescence quantum yields were determined by relative method
using fluorescein reference as previously described [17].
2.4. General procedure for preparation of probes test solutions
The stock solutions of tested compounds 4a-4b (1 mM) were pre-
pared in acetonitrile (MeCN). The stock solutions of amino acids (L-Cys,
L-Hcy, L-ACC, L-GSH, Gly, L-Glu, L-Lys, L-Met – 3 mM, L-Tyr – 2 mM), 2-
mercaptoethanol (ME, 3 mM), thioglycolic acid (TGA, 3 mM), sodium
bisulphite (NaHSO3, 3 mM), sodium hydrosulphide (NaSH, 3 mM), so-
dium sulfide (Na2S, 3 mM), hydrogen peroxide (H2O2, 3 mM) and N-
ethylmaleimide (30 mM) were prepared in distilled water. All of the
solutions were prepared immediately before the experiments. Typically,
the probes (20 μM) were incubated with an appropriate amount of a
stock solution of each analyte in a phosphate buffer (0.1 M, pH 7.4) at
room temperature (25 °C) for 15 min. MeCN was used as a cosolvent
(2%, v/v).
In these studies, an acetate buffer (0.1 M, pH 3.2–5.1), phosphate
buffer (0.1 M, pH 6.4–7.8) and carbonate-bicarbonate buffer (0.1 M, pH
9.4–10.4) were used. All of the pH measurements were made with a
CPI-551 microcomputer pH/ion meter (Elmetron, Poland). Each of the
experiments was performed in triplicate.
To the best of our knowledge, the probes 4a-4b are so far not
known.
The absorption and emission properties of xanthone (1), the ob-
tained probes 4a-4b and their strongly fluorescent precursors 3a-3b
were studied by means of UV-VIS and luminescence spectroscopy in
acetonitrile (MeCN) and in chloroform (CHCl3) solutions. The obtained
data are summarized in Tables 1. In addition, the absorption spectra of
the studied compounds in MeCN are presented in Fig. 1. Excitation-
emission matrix contour plots for 3a and 3b in MeCN are presented in
Fig. 2. The derivatives 4a-4b exhibit one absorption band in the UV
region located at 341–348 nm. Whereas, the fluorescent intermediate
Table 1
The spectroscopic data of compounds 3 and 4 in different media.
2.5. Determination of cell viability and studies of the response to thiols in
living cells
1
3a
3b
4a
4b
λabsa, nm
339
6200
388
6000
410
351
9400
8000
506
96
341
5900
342
6400
The human cell line HeLa was obtained from CBMM PAN Lodz. The
cell line was cultured in RPMI 1640 medium (Gibco) with 10% fetal
bovine serum (Gibco) supplemented with penicillin and streptomycin
antibiotic at 37 °C in 5% CO2 air atmosphere/95% air atmosphere. The
HeLa cells were seeded in 96-well plates at a concentration of 1 × 104
cells per well. The stock solutions of the probes 4a-4b (625 μM) were
prepared in DMSO.
The cytotoxicity of the compounds 4a-4b was determined using an
MTT-based assay according to the procedure described in Ref. [67]. The
cells were treated with the indicated concentrations (0–25 μM) of
compounds 4a-4b for 24 h.
To determine the response to thiols in living cells, the fluorescence
intensity was measured using a Synergy 2 microplate reader (BioTek,
Winooski, VT, USA) operated by the Gen5 program. For the control
experiment, cells in part of wells were pretreated with 400 μM of N-
ethylmaleimide and incubated at 37 °C for 20 min. A stock solution of
NEM (10 mM) was prepared in distilled water. Then the HeLa cells were
εa, M–1 cm–1
λema, nm
–
–
–
–
515
127
40
13.2
381
–
–
0.05
–
343
–
–
0.13
–
343
Stokes shifta, nm
a
Φem
34
,
%
τa, ns
11.2
404
346
6900
5300
520
116
45
λabsb, nm
342
εb, M–1 cm–1
9600
6700
7200
5600
λemb, nm
–
–
–
–
–
526
145
46
–
–
–
–
–
–
–
Stokes shiftb, nm
b
Φem
,
%
λabsc, nm
–
–
345
6100
348
6000
εc, M–1 cm–1
–
a
MeCN.
CHCl3.
b
c
H2O (with 2% MeCN, pH 7.4).
3

A. Grzelakowska, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry387(2020)112153
404–410 nm). The results indicate that the introduction of –NH2 groups
leads to a red shift in the absorption band compared with 9H-xanthen-
9-one (1), which is in accordance with the electronic effects that sub-
stituents can exert. Then, the presence of maleimide groups does not
cause any significant change in the absorption spectra.
2-aminoxanthone (3a) and 2,7-diaminoxanthone (3b) exhibit a
fluorescence emission band with the maximum located in the range
506–526 nm. The presence of additional amino group in the structure of
dye 3b causes a small blue shift of λem in comparison with the dye 3a.
The obtained results illustrate that dyes 3a-3b exhibit fluorescence with
an emission band characterized by a Stokes shift of ˜96 to 145 nm.
These values indicate that the geometries of the singlet excited states
differ from the geometries of the ground states. As can be seen from
data presented in Table 1, the fluorescence quantum yields (Φem) of
compounds 3a-3b ranges from 34% to 46%. The obtained fluorescence
intensity decays were fitted by monexponential model (Fig. S5). The
singlet lifetimes of 3a and 3b in MeCN are 13.2 ns and 11.2 ns, re-
spectively.
The replacement of amino group in compounds 3a-3b by a mal-
eimde moiety (4a-4b) causes a fluorescence quenching. The fluores-
cence reduction by attachment of the maleimide ring proceeds via a
photoinduced electron transfer (PET) mechanism [67]. The transfer of
an electron from the excited state of fluorophore to the LUMO of the
maleimide group results in decreased fluorescence due to non-radiative
relaxation. After the thiol addition to the double bond in maleimide
Fig. 1. Electronic absorption spectra of xanthone (1), compounds 3a-3b and
4a-4b in MeCN (50 μM).
3a exhibits one absorption band (λmax = 381–388 nm) and the ab-
sorption spectra of compound 3b shows two bands, with one band each
located in both the ultraviolet and visible ranges (λmax = 349–351 and
Fig. 2. Excitation-emission matrix contour plots for 3a (a) and 3b (b) in MeCN (20 μM).
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A. Grzelakowska, et al.
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Fig. 3. The fluorescence spectra of aqueous solutions of 20 μM 4a-4b, 0.1 M phosphate buffer (pH 7.4) and 2% MeCN obtained after the addition of 40 μM various
analytes and the incubation at 25 °C for 15 min (λex = 355 nm).
Fig. 5. Time-dependent fluorescence intensity of probes 4a-4b (20 μM) without
and in the presence of L-Cys (40 μM) in phosphate buffer (2% MeCN, pH 7.4).
The mixtures were incubated at 25 °C for 15 min (λex = 355 nm).
ground state and the fluorescence is restored [68,69].
It is evident that the solvent polarity has little influence on the
spectroscopic properties of the tested compounds. Despite different
solvent polarity, the position of maximum absorption band was prac-
tically the same. Thus, the solvatochromism was not observed.
3.2. Studies of the fluorescent response to thiols
Regarding the application of compounds 4a-4b as probes for thiols,
their spectroscopic properties upon the addition different analytes were
investigated.
It is obvious that the selectivity is crucial property of probes from an
application point of view. To determinate the selectivity of obtained
probes, the fluorescence spectra of compounds 4a-4b in aqueous solu-
Fig. 4. The fluorescence intensity measured at λem for the solutions of 20 μM
4a-4b, 0.1 M phosphate buffer (pH 7.4) and 2% MeCN without and in the
presence of 40 μM various analytes and upon the addition of additional
40 μM L-Cys. The mixtures were incubated at 25 °C for 15 min (λex = 355 nm).
tions (containing 2% MeCN as a cosolvent) in the presence of various
analytes were measured. Fig. 3 illustrates the fluorescence spectra of
4a-4b in the presence of different amino acids and other competitive
compounds. Additionally, fluorescence intensities of tested probes at
λem upon the addition of L-Cys in the presence of competitive com-
pounds are shown in Fig. 4. The products formed upon reaction of 4a
and 4b have intensive fluorescence with a single emission peaks at
469 nm and 474 nm, respectively. It can be seen that the addition of L-
moiety, the LUMO of the resulting succinimide group is higher in en-
ergy and the electron transfer from the fluorophore excited state is not
thermodynamically favored. Thus, relaxation occurs directly to the
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A. Grzelakowska, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry387(2020)112153
Fig. 6. The fluorescence spectra of aqueous solutions of 20 μM 4a-4b, 0.1 M phosphate buffer (pH 7.4) and 2% MeCN obtained after addition of 0–200 μM L-Cys. The
mixtures were incubated at 25 °C for 15 min (λex = 355 nm).
Fig. 7. The fluorescence intensity measured at λem for the solutions of 20 μM 4a-4b, 0.1 M phosphate buffer (pH 7.4) and 2% MeCN after the addition of 0–200 μM L-
Cys. The mixtures were incubated at 25 °C for 15 min (λex = 355 nm). Errors bars represent the standard deviations of three trials.
Cys to solution of compounds 4a-4b at a pH of 7.4 results in great in-
crease of their fluorescence intensity – over 25-fold in case of 4a and
over 60-fold in case of 4b. Moreover, the addition of other competitive
analytes does not affect the fluorescence response to L-Cys and the
probes selectively recognize L-Cys. However, the addition of L-Cys to
solutions of probe 4a and mercapto amino acids (L-Hcy, L-GSH and L-
ACC) causes a smaller increase in fluorescent intensity than in case of
the similar samples with 4b.
The results show that no significant changes were observed in the
absorption spectrum upon the addition different analytes (Figs. S6–S7).
The addition of L-Cys to solution of 4a-4b resulted in a small bato-
chromic shift of their λmax at a pH of 7.4.
Additionally, the time-dependent fluorescence intensity changes of
compounds 4a-4b in the presence of L-Cys were studied (Fig. 5). It is
obvious that the addition of L-Cys to solutions of 4a-4b resulted in an
increase in fluorescence intensity at λmax over time. In the presence of
40 μM L-Cys, the increase in fluorescence intensity of 4a-4b reaches a
plateau within 600 s. Thus, the fluorescence response of the tested
compounds to L-Cys was obtained in a short period of time.
Fig. 8. The dependence of the fluorescence intensity of probes 4a-4b (20 μM) at
The response sensitivity of probes 4a-4b to L-Cys was investigated
by fluorescence titration experiments in aqueous solutions (containing
2% MeCN as a cosolvent, pH 7.4). Fig. 6 displays the fluorescence
λ
em in the absence and in the presence of L-Cys (40 μM) on the pH values. The
mixtures were incubated at 25 °C for 15 min (λex = 355 nm).
6

A. Grzelakowska, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry387(2020)112153
Fig. 9. The fluorescence spectra of 4a (20 μM) and 4b (20 μM) in the absence and in the presence of L-Cys (3 mM) and a mixture of L-Cys with NEM (3 mM) in
phosphate buffer (2% MeCN, pH 7.4). The mixtures were incubated at 25 °C for 15 min (λex = 355 nm).
Scheme 2. The proposed reaction of studied compounds 4a-4b with L-Cys.
responses of tested probes toward different concentration of L-Cys. As
shown, the fluorescence intensities at 469 nm (4a) and 474 nm (4b)
increase gradually with increasing concentration of L-Cys (0–200 μM).
When L-Cys concentration was in the range of 0–20 μM, the fluores-
cence intensity of 4a was linearly proportional to thiol concentration,
while 4b kept a linear relationship with L-Cys concentration in the
range of 0–40 μM (Fig. 7). It should be emphasized that the increase of
the measured fluorescence intensity at λem was linear up to approxi-
mately 1:1 maleimide-thiol ratio, while further addition of L-Cys did not
cause additional increase of the product fluorescent intensity or its
decay. The reactions of 4a and 4b with L-Cys occur in a stoichiometric
manner.
Based on these studies, the detection limits of probes for L-Cys were
determined on the basis of the standard deviation of the response and
the slope [70]. The detection limits were calculated according to the Eq.
(3):
3.3σ
s
DL =
(3)
where σ is the standard deviation of the response and s is the slope of
the calibration curve. The estimate of σ was carried out based on the
standard deviation of the blank. The fluorescence spectra of dyes
without L-Cys were measured 11 times and the standard deviation of the
Fig. 10. The viability of the HeLa cells after treatment with compounds 4a-4b
for 24 h (determined using an MTT-based assay).
7

A. Grzelakowska, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry387(2020)112153
Fig. 11. Fluorescence response of compounds 4a-4b (0–25 μM) in the human cells HeLa (10,000 cells per 100 μl; the fluorescence intensity at 460/40 nm was
measured – λex = 360/40 nm).
blank measurements was determined. s was obtained from plot of
function fluorescence intensity of probe and L-Cys concentration.
The lowest concentration of L-Cys that can be detected by the pre-
sented fluorescent probes is 0.117 μM for 4a and 0.121 μM for 4b.
It is evident that, the fluorescent probes may be influenced by the
pH of the extracellular and intracellular milieu. Therefore, the effect of
pH on the fluorescent response of probes 4a-4b to L-Cys was in-
vestigated. Fig. 8 shows the variation in fluorescence intensity for tested
probes with and without L-Cys as a function of pH (3.2–10.4). In the
presence of L-Cys, fluorescence intensity increased in the pH range
above 5.1 to 9.4, but the highest increase in fluorescence intensity in
the presence of this amino acid was observed at a physiological pH
value (7.4). In the case of compounds 4a-4b without L-Cys, the fluor-
escence intensity remains almost unchanged in the same pH range. A
slight increase fluorescence intensity of 4a-4b above pH 8.0 was ob-
served. The specific behavior under more alkaline condition results
from the hydrolysis process in which N-substituted maleimides are in-
volved [28]. It can be seen from the obtained data that the investigated
compounds 4a-4b exhibit strong fluorescent response to L-Cys at phy-
siological pH values.
Furthermore, the emission spectra of probes 4a-4b were recorded in
the presence L-Cys which was previously treated with
N–ethylmaleimide (NEM – commonly used as thiol-blocking reagent)
[71]. As shown in Fig. 9, L-Cys treated with NEM did not enhance the
fluorescence of the studied compounds 4a-4b. This suggests that the
optical response toward L-Cys is the result of the reaction of the probes
with sulfhydryl group present in the structure of amino acid.
It is well known from the literature that thiols are capable of ad-
dition to the double bonds of unsaturated compounds to give various of
sulphides [72]. Typically, reactions involving nucleophilic addition of
thiol undergo through nucleophilic attack by their thiolate anion, Thus,
based on previously reported studies of the reaction mechanism be-
tween thiols and various maleimide derivatives [68,73–76], during the
reaction of probes 4a-4b and L-cysteine the products of the addition to
the double bonds in maleimide moiety are expected to be formed. The
proposed reactions between L-Cys and the obtained probes are shown in
Scheme 2. Moreover, an attempt was made to confirm the mechanism
of this reaction in the case of probes 4a-4b. The 1H NMR spectroscopic
analysis provides evidence for the addition of the thiol to the double
bond in maleimide moiety (Figs. S8 and S9). Upon addition of L-Cys to
the DMSO-d6 solution of probe 4a, the signal from the protons (CH
meleimide ring) at 7.08 ppm disappeared while new peaks at 4.26 ppm
and in the range of 2.50–3.00 ppm were observed. A similar relation-
ship was observed for probe 4b. In the presence of L-Cys, the signal at
7.61 ppm disappeared and new peaks at 4.26 ppm and in the range of
2.50–3.00 ppm appeared.
Finally, a cell culture study was used to evaluate the applicability of
compounds 4a-4b for the detection of thiols in biological environment.
To quantify the cytotoxicity of the tested compounds 4a-4b, an
MTT-based assay that measured the metabolic activity of viable cells,
was used. The human cell line HeLa was treated with various con-
centrations (0–25 μM) of the studied compounds. It was found that,
upon exposure to compounds 4a-4b for 24 h the majority of HeLa cells
remained viable (Fig. 10).
The addition of probes 4a-4b to HeLa cells resulted in an increase in
fluorescence intensity at 460/40 nm (λex = 360/40 nm) (Fig. 11).
Moreover, the increase in fluorescence was linearly proportional to 4a-
4b concentration (Figs. S10 and S11). In the wells where the cells were
pretreated with NEM and then incubated with the compounds 4a-4b
significant changes in fluorescence were not observed. The response
was similar to the untreated control cells.
The results presented in Fig. 11 confirmed that an increase in
fluorescence was caused by the reactions of the probes with thiols from
HeLa cell line milieu.
Additionally, the living cell imaging experiments were performed.
HeLa cells incubated with 4a-4b (25 μM) in culture media for 30 min
show blue fluorescence (Fig. 12D and H). The cells which were pre-
treated with NEM and the further incubated with 4a-4b in culture
media for 30 min showed non-fluorescence (Fig. 12F and J).
The obtained data demonstrated the studied probes 4a-4b can be
potentially used in living cell measurements.
4. Conclusions
The presented research has been focused on the synthesis of novel
fluorescent probes based on a xanthone skeleton. The compounds were
characterized by 1H NMR spectroscopy APCI mass spectrometry.
Spectroscopic studies of the presented xanthone derivatives under
various conditions clearly indicates that the tested compounds are good
candidates for potential application as “turn on” fluorescent probes for
8

A. Grzelakowska, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry387(2020)112153
Fig. 12. Images of the HeLa cells: (A) bright-field and (B) fluorescence microscopic images of HeLa cells (control); (C, G) bright-field and (D, H) fluorescence
microscopic images of HeLa cells after incubated with probes 4a-4b (25 μM) for 30 min; (E, I) bright-field and (F, J) fluorescence microscopic images of HeLa cells
pretreated with NEM (400 μM) for 30 min and then incubated with probes 4a-4b (25 μM) for 30 min.
the detection of sulphydryl compounds, especially L-cysteine. Reaction
of 2-maleimidoxanthone (4a) and 2,7-dimaleimidoxanthone (4b) with
L-Cys leads to the formation of the fluorescent products under physio-
logical conditions (pH 7.4). Fluorescence intensities of probes 4a and
4b have greatly enhanced 25-fold and 60-fold, respectively. The reac-
tions of 4a and 4b with L-Cys occur in a stoichiometric manner and the
lowest concentration of L-Cys that can be determined by the presented
probes is 0.117–0.121 μM. As a proof of the concept of the usefulness of
the probes 4a-4b for the detection of thiols in cellular milieu, the
fluorescent response of these compounds to thiols in HeLa cell line was
investigated. The incubation of HeLa cells with the probes 4a-4b re-
sulted in the increase fluorescence, which was inhibited by N-ethyl-
maleimide. The presented novel derivatives of xanthone have potential
application as fluorescent probes for L-Cys and can be an alternative to
commonly used N-substituted maleimides.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
9

A. Grzelakowska, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry387(2020)112153
influence the work reported in this paper.
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
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