Long-wavelength TCF-based fluorescence probes for the detection and intracellular imaging of biological thiols

Article abstract of DOI:10.1039/c8cc01661e

Two 'turn on' TCF-based fluorescence probes were developed for the detection of biological thiols (TCF-GSH and TCFCl-GSH). TCF-GSH was shown to have a high sensitivity towards glutathione (GSH) with a 0.28 μM limit of detection. Unfortunately, at higher GSH concentrations the fluorescence intensity of TCF-GSH decreased and toxicity was observed for TCF-GSH in live cells. However, TCFCl-GSH was shown to be able to detect GSH at biologically relevant concentrations with a 0.45 μM limit of detection. No toxicity was found for TCFCl-GSH and a clear 'turn on' with good photostability was observed for the exogenous addition of GSH, Cys and HCys. Furthermore, TCFCl-GSH was used to evaluate the effects of drug treatment on the levels of GSH in live cells.

Full text of DOI:10.1039/c8cc01661e

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Long‐wavelength TCF‐based fluorescence probes for the detection
and intracellular imaging of biological thiols
a
a
b
c
Adam C. Sedgwick *, Jordan E. Gardiner , Gyoungmi Kim , Maksims Yevglevskis , Matthew D.
Received 00th January 20xx,
Accepted 00th January 20xx
c
a
a
b
a
Lloyd , A. Toby A. Jenkins , Steven D. Bull *, Juyoung Yoon * and Tony D. James *
DOI: 10.1039/x0xx00000x
www.rsc.org/
Two ‘turn on’ TCF‐based fluorescence probes were developed for With our research, we are interested in the development of
the detection of biological thiols (TCF‐GSH and TCFCl‐GSH). TCF‐GSH reaction based fluorescent probes for the detection of
was shown to have a high sensitivity towards glutathione (GSH) biologically relevant species to be used as powerful tools for the
with a 0.28 µM limit of detection. Unfortunately, at higher GSH understanding of diseases or diagnostics.9
‐13
Currently, a
concentrations the fluorescence intensity of TCF‐GSH decreased number of fluorescent probes exist for the detection of
and toxicity was observed for TCF‐GSH in live cells. However, TCFCl‐ biological thiols.1
4‐20
However, long excitation/emission
GSH was shown to be able to detect GSH at biological relevant wavelength fluorescent probes are highly desirable as they
concentrations with a 0.45 µM limit of detection. No toxicity was allow deeper tissue penetration, minimal background auto‐
found for TCFCl‐GSH and a clear ‘turn on’ with good photostability fluorescence from proteins and photodamage to the biological
was observed for exogenous addition of GSH, Cys and HCys. samples. Therefore, in this work we looked to develop TCF‐
11
Furthermore, TCFCl‐GSH could be used to evaluate the effects of based systems for the long wavelength detection of GSH.
drug treatment on the levels of GSH in live cells.
TCF‐based fluorophores have an internal charge transfer (ICT)
donor‐π‐acceptor (D‐π‐A) structure with long emission
wavelengths. (See ESI – Scheme S1) As a result, TCF
fluorophores have been used in many applications such as non‐
linear optic chromophores and fluorescent probes.
Hilderbrand et al. previously developed a ‘turn on’ sulfonamide
Glutathione (GSH), Cysteine (Cys) and Homocysteine (HCys)
play
homeostasis.
a vital role in maintaining the biological redox
1, 2
GSH is a natural tripeptide (γ‐L‐glutamyl‐L‐
21‐25
cysteinyl‐glycine), which exists in the thiol reduced form (GSH)
2
and disulphide‐oxidised (GSSG) form. GSH is the predominant
based TCF fluorescent probe for the detection of biological
form, which exists in millimolar concentrations in most cells
26
thiols. However, a PEG unit was required to provide aqueous
3
where it functions as an antioxidant. Elevated levels of GSH are
solubility and cell permeability. The probe was successfully
shown to detect biological thiols in 3T3 cells. We believed the
synthesis of the analogous sulfonate ester would overcome the
need for a PEG unit and provide a much simpler synthesis. The
TCF fluorophore unit was synthesised as previously reported
using the reaction of 3‐hydroxy‐3‐methyl‐2‐butanone,
malonitrile and NaOEt in EtOH. With the TCF unit in hand, the
common in the presence of oxidative stress and the
susceptibility of a cell towards reactive oxygen or nitrogen
species (ROS/RNS) largely depends on the concentration of
intracellular GSH.4 Therefore, the change in the level of GSH
concentration has been associated with a number of diseases
such as AIDS, liver damage, cancer and neurodegenerative
disease (Alzheimer’s disease).6 Interestingly, it was reported
‐7
, 7
(
D‐π‐A) systems TCF‐OH and TCFCl‐OH were isolated in high
that at early stages of cell proliferation (S, G
2
and M phases),
27
yield using microwave reaction conditions. The TCF phenols
were then reacted with 2,4‐dinitrobenzenesulfonylchloride to
afford the desired fluorescent probes TCF‐GSH and TCFCl‐GSH
in satisfactory yields (55 % and 64 %) (Fig. 1)
GSH was found to localise at the nucleus. This was believed to
prevent apoptosis and provide a reduced environment for
transcription factors to bind to DNA.8
a.
Department of Chemistry, University of Bath, Bath, BA2 7AY, UK. Email:
t.d.james@bath.ac.uk; s.d.bull@bath.ac.uk
Department of Chemistry and Nano Science, Ewha Womans University,
b.
Seoul 120‐750, Korea. E‐mail: jyoon@ewha.ac.kr
c
Drug & Target Development, Department of Pharmacy & Pharmacology, University
of Bath, Claverton Down, Bath, BA2 7AY, UK.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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Sadly, despite TCF‐GSH being sensitive towards GSH, we only
observed a clear ‘off‐on’ response for the exogenous addition
of Cys in HeLa cells. Furthermore, TCF‐GSH was shown to have
toxicity in a cell viability experiments (See ESI – Fig S16 and S17).
TCF‐GSH is therefore unsuitable for cell imaging experiments
for the detection of biothiols. Interestingly, cellular imaging
experiments did not require any additional additives to
compensate for the 20 % v/v DMSO required in the in vitro
experiments.
We therefore turned our attention towards the fluorescence
properties of TCFCl‐GSH. In order to produce a fluorescence
response, TCFCl‐GSH also required pH 8.0 buffer solution (20 %
DMSO). However, TCFCl‐GSH was shown to be less sensitive
towards the biological thiols and no decrease in fluorescence
intensity was observed at higher concentrations (Fig 3).
Fig. 1 – TCF‐based fluorescent probes for the detection of biological thiols (TCF‐
GSH and TCFCl‐GSH).
On the addition of GSH both probes TCF‐GSH and TCFCl‐GSH
change colour from yellow to purple. (See ESI – Fig S1 and S2)
We evaluated the fluorescence behaviour of TCF‐GSH, in pH 8.0
buffer solution (20 % v/v DMSO). (See See ESI – Fig S3 and S4)
Interestingly, 20 % v/v DMSO was required for the reaction
between the probe and the chosen biological thiol to take place.
We then evaluated TCF‐GSH for the detection of GSH, given that
it is the most predominant biological thiol in cells. Remarkably,
TCF‐GSH was very sensitive towards GSH producing a full ‘turn
on’ fluorescence response in the presence of 25 µM GSH.
Unfortunately, at concentrations > 50 µM the fluorescence
intensity of TCF‐GSH began to drop dramatically. This is due to
600000
GSH
20
15
10
5
0
500000
4
00000
00000
3
0
100
200
300
400
500
600
700
800
Concentration (GSH)/ micromolar
28
200000
attack of the TCF fluorophores by nucleophiles. (See ESI – Fig
S5 – S11).
100000
0
600
620
640
660
680
700
720
Wavelength/ nm
1
1
20000
00000
1
1
6
4
Fig. 3 ‐ Fluorescence spectra of TCFCl‐GSH (5 µM) with addition of GSH (0 – 750 µM) and
o
15 min wait between additions in PBS buffer solution, 20 % DMSO, pH 8.00 at 25 C. λex
12
GSH
1
0
8
6
4
2
0
= 560 ± 15 nm
80000
60000
40000
20000
0
We then evaluated the selectivity of TCFCl‐GSH towards other
biologically relevant thiols and amino acids (See ESI – Fig S14
and S15). As for TCF‐GSH, TCFCl‐GSH reacted with the R‐SH
containing amino acids Cys and HCys. While excellent selectivity
for GSH was observed against other amino acids. This permitted
the evaluation of TCFCl‐GSH for the detection of exogenous and
endogenous thiols in live cells. As shown in Fig. 4(See ESI – Fig
S18 and S19 for TCF‐GSH), TCFCl‐GSH displayed an already
strong fluorescence response in live cells Fig 4 ‐ (a). This
0
100
200
300
400
500
Concentration (GSH)/ micromolar
6
00
620
640
660
680
700
720
Wavelength/ nm
Fig. 2 ‐ Fluorescence spectra of TCF‐GSH (5 µM) with addition of GSH(0 – 500 µM) and observation was due to the presence of endogenous thiols
o
1
=
5 min wait between additions in PBS buffer solution, 20 % DMSO, pH 8.00 at 25 C. λex
560 ± 15 nm. Orange dashed lines indicate fluorescence decrease at high GSH
reacting with TCFCl‐GSH. However, pre‐treatment of HeLa cells
with the thiol reactive N‐methylmaleimide (NMM) led to the
reduction of endogenous thiols and therefore low fluorescence
intensity was observed when TCFCl‐GSH was added Fig 4 ‐ (b).
The addition of 200 µM of an exogenous thiol (Cys, HCys, or
GSH‐Methyl ester) led to a clear change in fluorescence
intensity demonstrating the ability of TCFCl‐GSH to detect thiols
in cells.
concentrations.
We then evaluated the selectivity of TCF‐GSH towards other
biologically relevant thiols and amino acids (See ESI – Fig S12
and S13). As predicted, TCF‐GSH reacted with the other
sulphydryl (R‐SH) compounds, Cys and HCys with Cys producing
the largest fluorescent response. However, the overall
We then evaluated the ability of TCFCl‐GSH to detect changes
in the concentration levels of endogenous thiols through the
addition of drugs and reactive oxygen species (ROS) such as
concentrations of both Cys and HCys are low in comparison to
GSH in cells.2
9, 30
TCF‐GSH demonstrated an excellent selectivity
for GSH against other amino acids. This excellent selectivity
permitted the evaluation of TCF‐GSH for the detection of
exogenous and endogenously generated thiols in live cells.
H
2
2
O . It is well known that GSH protects against drug induced
toxicity and acts as a ROS scavenger. Therefore in Fig. 5 and 6
(
2 2
See ESI – Fig S20 and S21 for TCF‐GSH), the addition of H O
2
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500 µM) or Cisplatin (200 µM) resulted in the depletion of the
COMMUNICATION
(
endogenous thiols and consequently reduced fluorescence was
observed when TCFCl‐GSH was added. Subsequently, the
3
1
addition of the GSH producing drug N‐Acetylcysteine
recovered the GSH levels resulting in a large increase in
fluorescence intensity.
Fig. 6 ‐ Intracellular fluorescence change caused by drug treatment. (B) HeLa cells were
incubated with 200 μM cisplatin with or without 2 mM NAC for 6 hr and stained with
2
0 μM TCFCl‐GSH for 20 min. Quantitative data of Fluorescence intensity was
calculated by FV10‐ASW 4.0 software and measured per one cell. Results are
expressed as mean ± standard deviation of three independent experiments. Ex. 559
nm/em. 575 – 675 nm. Scale bar: 20 μm.
In summary, two ‘turn on’ TCF‐based fluorescent probes have
been developed for the detection of biological thiols (TCF‐GSH
and TCFCl‐GSH). TCF‐GSH was shown to have a high sensitvity
towards glutathione (GSH). Unfortunately, at higher GSH
concentrations the fluorescence intensity of TCF‐GSH
decreased and toxicity was observed in live cells making it
Fig. 4 ‐ Fluorescence imaging in live cells. HeLa cells were preincubated with 0.2 mM unsuitable for cellular imaging. However, TCFCl‐GSH was shown
NMM for 20 min and washed with Dulbecco's phosphate‐buffered saline (DPBS) and
incubated with 200 μM cysteine, homocysteine and GSH‐MEE for 20 min. After washing
Also, no toxicity was observed for TCFCl‐GSH and a clear ‘turn
with DPBS, cells were stained with 20 μM TCFCl‐GSH for 20 min and fluorescence images
to be able to detect GSH at biological relevant concentrations.
on’ response was observed upon the exogenous addition of
acquired by confocal microscopy. (a) only TCFCl‐GSH, (b) NMM + TCFCl‐GSH, (c) NMM +
cysteine + TCFCl‐GSH, (d) NMM + homocysteine + TCFCl‐GSH and (e) NMM + GSH‐MEE GSH, Cys and HCys. Furthermore, TCFCl‐GSH was able to
+
TCFCl‐GSH. Top: fluorescence image (ex. 559 nm/em. 575‐675 nm), bottom: merged
evaluate the effects of drug treatment and the addition of ROS
) on live cells both of which resulted in a depletion of
cellular GSH levels and a reduced fluorescence intensity.
Subsequent, addition of NAC increased the GSH levels and
enhanced the observed fluorescence intensity.
image with DIC. Scale bar. 20 μm. Quantitative data of Fluorescence intensity was
calculated by FV10‐ASW 4.0 software and measured per one cell. Results are expressed
as mean ± standard deviation of three independent experiments.
(H
2
O
2
,
Acknowledgements
We would like to thank the EPSRC, the University of Bath and
Prostate Cancer UK (PG14‐009) for funding. ACS and JEG thank
the EPSRC for studentships. TDJ wishes to thank the Royal
Society for
a Wolfson Research Merit Award. NMR
characterisation facilities were provided through the Chemical
Characterisation and Analysis Facility (CCAF) at the University of
Bath (www.bath.ac.uk/ccaf). The EPSRC UK National Mass
Spectrometry Facility at Swansea University is thanked for
analyses. JY thanks the support from the National
control
H
2
O
2
2 2
H O + NAC
Research Foundation of Korea (NRF), which was funded by the
Korea government (MSIP) (No. 2012R1A3A2048814). ACS, MY,
Fig. 5 ‐ Intracellular fluorescence change caused by drug treatment. (A) HeLa cells were
incubated with 500 μM H2O2 with or without 2 mM NAC for 6 hr and stained with 20 MDL and TDJ are members of the Cancer Research @ Bath
μM TCFCl‐GSH for 20 min. Quantitative data of Fluorescence intensity was calculated by (CR@B) network. All data supporting this study are provided as
FV10‐ASW 4.0 software and measured per one cell. Results are expressed as mean ±
standard deviation of three independent experiments. Ex. 559 nm/em. 575 – 675 nm.
Scale bar: 20 μm.
supplementary informaꢀon accompanying this paper (ESI†).
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