A Native-Chemical-Ligation-Based Turn-on Fluorescent Probe for Selective Detection of Cysteine

Article abstract of DOI:10.1002/jccs.201500466

Selective and quantitative detection of biological thiols such as cysteine, homocysteine, and glutathione is often necessary because abnormal levels of such thiols can cause some diseases. Here, we report that bis(pentafluorophenyl) 1,4-benzenedicarbothionic acid diester can serve as a turn-on fluorescent probe for selective detection of cysteine vis-a-vis homocysteine and glutathione. When cysteine was added to a mixture of the diester and a sodium phosphate buffer solution with THF (60 vol%), which is non-fluorescent, the mixture became green-fluorescent. In contrast, addition of homocysteine or glutathione did not make the mixture fluorescent. A native-chemical-ligation-based mechanism is proposed.

Full text of DOI:10.1002/jccs.201500466

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Invited Paper
A Native-Chemical-Ligation-Based Turn-on Fluorescent Probe for Selective Detection
of Cysteine
Masaki Shimizu,* Hiroki Fukui and Ryosuke Shigitani
Faculty of Molecular Chemistry and Engineering, Kyoto Institute of Technology, 1 Hashikami-cho, Matsugasaki,
Sakyo-ku, Kyoto 606-8585, Japan
(Received: Nov. 17, 2015; Accepted: Jan. 7, 2016; Published Online: Feb. 22, 2016; DOI: 10.1002/jccs.201500466)
Selective and quantitative detection of biological thiols such as cysteine, homocysteine, and glutathione is
often necessary because abnormal levels of such thiols can cause some diseases. Here, we report that
bis(pentafluorophenyl) 1,4-benzenedicarbothionic acid diester can serve as a turn-on fluorescent probe
for selective detection of cysteine vis-a-vis homocysteine and glutathione. When cysteine was added to a
mixture of the diester and a sodium phosphate buffer solution with THF (60 vol%), which is non-fluores-
cent, the mixture became green-fluorescent. In contrast, addition of homocysteine or glutathione did not
make the mixture fluorescent. A native-chemical-ligation-based mechanism is proposed.
Keywords: Fluorescence; Sensing; Thiols.
Masaki Shimizu was born in 1965 in Tokyo, Japan. He received his Ph.D. from the Tokyo Institute of
Technology in 1994 under the supervision of Profs. Takeshi Nakai and Koichi Mikami. After working
at Mitsubishi Chemical Co. Ltd. for 14 months, he joined Prof. Tamejiro Hiyama’s group at the Re-
search Laboratory of Resources Utilization, Tokyo Inst. of Tech. as an Assistant Professor in 1995. In
1998, he moved to Kyoto University as an Assistant Professor. He spent one year (1999~2000) at
Massachusetts Institute of Technology as a postdoctoral fellow in the group of Prof. Stephen L.
Buchwald and was promoted to Associate Professor of Kyoto Univ. in 2003. In 2012, he became Pro-
fessor at Kyoto Institute of Technology. His research interest focuses on design and invention of func-
tional p-conjugated molecules as well as the development of synthetic methodologies directed toward
functional organic materials.
* Corresponding author. Tel: +81-75-724-7830; Fax: +81-75-724-7806; Email: mshimizu@kit.ac.jp
Supporting information for this article is available on the www under http://dx.doi.org/10.1002/jccs.201500466
J. Chin. Chem. Soc. 2016, 63, 317-322
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INTRODUCTION
Biological thiols such as cysteine (Cys), homocy-
steine (Hcy), and glutathione (GSH) are necessary for vari-
ous biological processes, and a surplus or deficiency of
thiols in the human body can cause some diseases. Hence,
accurate monitoring of biothiol levels is useful, and a vari-
ety of colorimetric and fluorescent probes for detection of
biothiols have been developed to date.1 The sensing me-
chanism of such probes is generally triggered by a nucle-
ophilic attack on an electrophilic functional group under
strong basic conditions or by coordination with a metal of
sulfhydryl groups. Such approaches make it difficult to dis-
criminate Cys and Hcy because their molecular structures
are similar, as is their chemical reactivity. Therefore, highly
selective detection of either Cys or Hcy is one of the chal-
lenging problems in the field of biothiol sensing.2
Fig. 1. Molecular structures of 1,4-bis(carbonyl)-2,5-
Native chemical ligation (NCL) is widely used as a
versatile method for protein backbone synthesis and in-
volves transthioesterification of a peptide a-thioester with
a peptide containing an N-terminal cysteine, followed by
facile rearrangement of the acyl group of the resulting
thiocarbonyl moiety from a sulfur to a nitrogen of the
cysteine moiety.3 Because of the mild conditions, high
chemoselectivity, and biocompatibility, NCL represents an
attractive mechanism for sensing of bioactive materials. On
the other hand, real examples of NCL-based probes are
scarce.4 Hence, it would be worthwhile to develop NCL-
based probes for selective detection of biological thiols.
We previously developed dimethyl 2,5-bis(diorgano-
amino)terephthalates (1) as novel fluorophores with green-
to-yellow emission in solution and in the solid state with a
moderate or high quantum yield, respectively (Figure 1).5
The excited state was confirmed to be caused by the intra-
molecular charge transfer (ICT) from the diorganoamino
group to methoxycarbonyl group, and the luminescent
properties depend on the degree of ICT, in other words, on
the combination of the donor and acceptor.6 Accordingly,
luminescent properties of thioesters 2 and amides 3 are ex-
pected to be largely different because they possess the same
electronic structure as that of compound 1 (Figure 1). Be-
cause thioesters and the corresponding amides can serve as
a starting material and a product of NCL, as stated above,
we hypothesized that sensing of biological thiols by means
of thioesters 2 as a probe is possible via monitoring of lumi-
nescent properties of this system. We were able to prove the
hypothesis: here, we report selective sensing of Cys by
2,5-bis(diphenylamino)-1,4-benzenedicarbothionic acid
diaminobenzenes 1–3.
bis(pentafluorophenyl)ester (Figure 1, 2a).
RESULTS AND DISCUSSION
Dithioester 2a was prepared in a high yield from
dimethyl 2,5-bis(diphenylamino)terephthalate (1: R = Ph)
via three steps involving hydrolysis, conversion to an acid
chloride, and esterification with C6F5SH in the presence of
pyridine as a base (Scheme 1). Purification of the crude
product by silica gel column chromatography gave 2a as a
red solid with good thermal stability (decomposition point:
234 °C).
Preparation of dithioester 2a.
Scheme 1
318
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Fluorescent Probe for Cysteine Sensing
In solution, 2a was non-fluorescent. In contrast, a
powder of 2a showed red fluorescence with an emission
maximum at 642 nm and a quantum yield of 0.21. Thus, 2a
exhibited aggregation-induced emission (AIE).7 Then, we
tested the AIE behavior of 2a in a mixture of THF and so-
dium phosphate buffer (PB; 10 mM, pH 7.2). If PB consti-
tuted up to 40 vol%, the mixture remained non-fluorescent
(Figure 2). When the aqueous fraction constituted more
than 40 vol%, the mixture became red-fluorescent with the
emission maximum near 615–620 nm, indicating that 2a
started to form aggregates (Figure S1 in Supporting Infor-
mation). The fluorescence intensity was enhanced as the
buffer’s proportion increased and reached a maximum at
80 vol% PB. According to these results, further experi-
ments of thiol sensing using 2a were performed with a
mixture of THF (60 vol%) and PB (40 vol%).
of 0–1 mM (R2 = 0.9824). The limit of detection of Cys was
found to be 58 mM on the basis of the signal-to-noise ratio
(S/N = 3). The concentration of Cys in serum or plasma of
healthy people is typically ~250 mM.8 Thus, the sensitivity
of 2a is suitable for detection of a Cys deficiency although
further improvements are necessary to achieve faster sens-
ing and better sensitivity.
To examine the sensing selectivity of 2a, Hcy and
GSH were subjected to the same conditions as those for
Cys sensing. As shown in Figure 5, no fluorescence of the
When an excess amount of Cys was added to a solu-
tion of 2a in a mixture of THF and PB (60/40), which was
non-fluorescent, the resulting solution showed green fluo-
rescence with the emission maximum at 521 nm upon exci-
tation at 390 nm (Figure 3). The fluorescence intensity in-
creased with time up to 10 h, and significant enhancement
was confirmed at 2 h after the addition of Cys. Thus, the re-
action time was set to 2 h in further experiments.
Fig. 3. Time-dependent fluorescence spectra of 2a (50
mM) in the presence of Cys (3 mM) in phos-
phate buffer (10 mM, pH 7.2, a mixture with
THF, the latter at 60 vol%) upon excitation at
390 nm at 37 °C. Inset: the fluorescence inten-
sity at 521 nm as a function of time.
To evaluate the sensitivity of 2a to Cys, fluorescence
spectra of a mixture of 2a and Cys in THF/PB (60/40) were
recorded at various concentrations of Cys. As shown in
Figure 4, the fluorescent signal was enhanced as the Cys
concentration increased (see Figure S2 in Supporting In-
formation), and the fluorescence intensity at 521 nm was
linearly proportional to the Cys concentration in the range
Fig. 4. Fluorescence spectra of 2a (50 mM) after 2 h
upon the addition of Cys at various concentra-
tions (0.1–5 mM) in phosphate buffer (10 mM,
pH 7.2, a mixture with THF, the latter at 60
vol%) upon excitation at 390 nm at 37 °C. Inset:
fluorescence intensity at 521 nm as a function
of Cys concentration.
Fig. 2. Fluorescence spectra of 2a (50 mM) in a mix-
ture of THF and a phosphate buffer at different
proportions of the buffer upon excitation at 390
nm.
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Proposed mechanism of Cys detection with
2a. The left pentafluorophenylthiocarbonyl
moieties of 2a, 3a, and 4–6 are omitted for
clarity.
Scheme 2
Fig. 5. Fluorescence intensity of 2a (50 mM) at 521 nm
in the presence of Cys, Hcy, or GSH (3 mM) in
phosphate buffer (10 mM, pH 7.2, a mixture
with THF, the latter at 60 vol%) upon excitation
at 390 nm at 37 °C.
resulting mixture was observed even 12 h after the addition
of Hcy or GSH (see also Figure S3 in Supporting Informa-
tion).9 Thus, 2a was confirmed to be a Cys-selective fluo-
rescent probe.
The proposed mechanism of the reaction of 2a with
Cys is shown in Scheme 2. First, transthioesterification
takes place between 2a and Cys to generate 4 (n = 1) with
releasing C6F5SH.10 After that, the amino group derived
from Cys undergoes intramolecular carbonyl addition thus
forming thiazolidine intermediate 5, in which the car-
bon–sulfur bond can be cleaved by the lone pair of the oxy-
gen attached to the carbon, giving rise to green-fluorescent
amide 3a,4 whose formation was confirmed by matrix-as-
sisted laser desorption ionization time-of-flight (MALDI-
TOF) mass spectrometry of the resulting solution (see Fig-
ure S5 in Supporting Information). Meanwhile, when Hcy
was added to 2a in THF/PB (60/40), the absorption spec-
trum of the resulting mixture became different from that of
2a (see Figure S6 in Supporting Information), indicating
that the initial transthioesterification took place success-
fully as in the case of Cys. The reaction of 2a with Hcy did
not produce green-fluorescent species (Figure 5) probably
because formation of tetrahydro-1,3-thiazine 6 is impossi-
ble due to the greater steric repulsion between the di-
phenylamino group and the 6-membered ring at the ortho-
position.2i,2l
lectively detects Cys but not Hcy and GSH because NCL-
based probes reported so far cannot discriminate Cys and
Hcy well. The topics of further research include enhance-
ment of the sensitivity, achievement of a quick response,
and application to sensing in live cells.
In summary, we designed and characterized bis(pen-
tafluorophenyl) 1,4-benzenedicarbothionic acid diester as
a turn-on fluorescent probe for highly selective sensing of
Cys on the basis of NCL. It is remarkable that the probe se-
EXPERIMENTAL
General: Melting point was determined using Seiko In-
strument Inc. TG/DTA6200. 1H NMR spectrum was measured on
a Bruker Avance II 300 (300 MHz) spectrometer. The chemical
320
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J. Chin. Chem. Soc. 2016, 63, 317-322

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Fluorescent Probe for Cysteine Sensing
1
shifts of H NMR are expressed in parts per million downfield
(dd, J = 8.1, 7.5 Hz, 8H), 7.50 (s, 2H); 13C NMR (CDCl3, 100
MHz): d 123.5, 123.8, 129.5, 130.1, 137.6, 142.2, 146.9, 184.5;
19F NMR (CDCl3, 282 MHz): d –162.5 – –162.3 (m, 4F), –150.9
(tt, J = 19.7, 2.8 Hz, 2F), –132.0 – –131.9 (m, 4F). IR (KBr): 3433,
3036, 1701, 1589, 1514, 1391, 1279, 1260, 1171, 1094, 982, 856,
756, 694 cm–1. HRMS–FAB (m/z): [M]+ calcd for C44H22O2N2F10S2
864.0963; found, 864.0956.
relative to the internal chloroform (d = 7.26 ppm). Splitting pat-
terns are indicated as s, singlet; d, doublet; m, multiplet. 13C NMR
spectrum was measured on Varian Mercury 400 (100 MHz) spec-
trometer with tetramethylsilane as an internal standard (d = 0
ppm) or chloroform-d (d = 77.0 ppm). 19F NMR spectrum was
measured on a Bruker Avance II 300 (282 MHz) spectrometer
with C6F6 as an internal standard (d = –163.7 ppm). Chemical
shift values are given in parts per million downfield relative to the
internal standards. Infrared spectrum (IR) was recorded on a
Shimadzu FTIR-8400 spectrometer. FAB-MS analysis was per-
formed with a JEOL JMS-700 spectrometer. TLC analysis was
performed by means of Merck Kieselgel 60 F254. Silica gel co-
lumn chromatography was carried out using Merck Kieselgel 60
(230–400 mesh). Reagent-grade dichloromethane was passed
through two packed columns of neutral alumina and copper oxide
under a nitrogen atmosphere before use. Tetrahydrofuran used for
UV–vis absorption and fluorescence measurements was pur-
chased from Kanto Chemical Co., Inc. and degassed with argon
before use. UV–vis absorption and fluorescence spectra were
measured with a Shimadzu UV–2550 spectrometer and Shimadzu
RF–5300PC spectrometer, respectively.
ACKNOWLEDGEMENTS
The authors thank Prof. Jong Seung Kim (Korea Uni-
versity) for giving us a reprint of his review article on lumi-
nescent and colorimetric chemosensors for detection of
thiols. One of the authors (M.S.) expresses his deepest ap-
preciation to Prof. Yi-Chou Tsai (National Tsing Hua Uni-
versity) for a valuable discussion during his stay in Taiwan
as a Visiting Lecturer of the Chemistry Promotion Center.
This work was supported by JSPS KAKENHI Grant Num-
bers 15H03795, 15H00996, 15H00740, 15K13671.
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Synthesis of 2a: A 100 mL two-necked flask was charged
with dimethyl 2,5-bis(diphenylamino)terephthalate (1.98 g, 3.74
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