Orchestration of dual cyclization processes and dual quenching mechanisms for enhanced selectivity and drastic fluorescence turn-on detection of cysteine

Article abstract of DOI:10.1039/c6cc09336a

We reported a new approach to achieve enhanced selectivity with a drastic turn-on fluorescence response for the detection of Cys through dual intramolecular cyclization processes and dual PET and ICT quenching mechanisms by the incorporation of an acrylate and a maleimide group onto two opposite sides of a single coumarin fluorophore.

Full text of DOI:10.1039/c6cc09336a

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc
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3
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DOI: 10.1039/C6CC09336A
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Orchestration of dual cyclization processes and dual quenching
mechanisms for enhanced selectivity and drastic fluorescence
turn-on detection of cysteine
Received 00th January 20xx,
Accepted 00th January 20xx
Hongjuan Tong, Jianhong Zhao,a,b Xiangmin Li, Yajun Zhang, Shengnan Ma, Kaiyan Lou* and
a,b
b
b
b
b
b,c
DOI: 10.1039/x0xx00000x
Wei Wang*
www.rsc.org/
We reported a new approach to achieve enhanced selectivity with advantage of reaction kinetic differences in the formation of
drastic turn-on fluorescence response for detection of Cys through different sizes of rings when the thiol and amine groups in Cys,
dual intramolecular cyclization processes and dual PET and ICT Hcy, and GSH undergo cascade nucleophilic reactions with a
quenching mechanisms by incorporation of an acrylate and a single recognition group. Such reactive groups involving a
1
1, 22
23
24
maleimide group onto two opposite sides of a single coumarin cyclization process include aldehyde,
fluorophore.
acrylate, thioester,
25
26-28
maleimide, or electron-deficient aromatic halide
(Scheme
S1, ESI†). Although good to excellent selectivity could generally
be achieved between Cys/Hcy and GSH, only modest selectivity
could be realized between Cys and Hcy due to their minute
structural difference by just one methylene group. The
selectivity was thus limited by the reaction kinetic differences in
the formation of x-membered ring for Cys and (x+1)-membered
ring for Hcy (x=5, 6, or 7 for recognition groups shown in Scheme
S1, ESI†) and difficult to improve for a single recognition group.
Here we reported a new approach to improve the selectivity
of Cys detection over Hcy by a synergetic use of two different
recognition groups with different intramolecular cyclization
processes. To demonstrate the feasibility of this approach, we
chose the acrylate group and the maleimide group in our probe
design as both the cascade reaction and sensing mechanisms
for the two groups toward aminothiols were well-studied
Low molecular weight aminothiols, such as cysteine (Cys),
homocysteine (Hcy), and glutathione (GSH), play different
important roles in biological systems.1 Among these
bioaminothiols, Cys is a precursor for peptides and proteins as
well as for GSH, acetyl coenzyme A, and taurine. The levels of
these aminothiols including Cys are highly regulated under
normal physiological conditions, while altered levels often
indicate different disease states.7-10 Therefore, methods for
detection these aminothiol’s levels with high selectivity under
complex biological context are greatly needed for studying their
specific physiological and pathological roles. So far, high
-6
1
1
performance liquid chromatography (HPLC),
capillary
UV-vis
12
13
electrophoresis,
electrochemical
assay,
1
4
15
spectroscopy, and fluorescence spectroscopy have been
employed in thiol detections. Among them, fluorescence
sensing has attract most attention recently because of its high
sensitivity, ease of use, and real-time imaging capabilities.16-18
Many fluorescent probes for Cys, Hcy and GSH have been
devised using various specific reaction and recognition
(Scheme 1). Acrylate group reacting with Cys/Hcy follows a
thiol-Michael addition and cyclization-deprotection cascade
23
process. The cyclization is 7-exo-trig for Cys and 8-exo-trig for
29
Hcy according to Baldwin’s nomenclature. While for the
maleimide group, a thiol-Michael addition and transcyclization
15, 19-21
groups.
To achieve selectivity, different cyclization
cascade reaction happens for Cys and Hcy in 6-exo-trig and 7-
processes were incorporated in the probe design, which take
25
exo-trig, respectively. We expected that the combined use of
the two different cyclization would give a better selectivity for
Cys over Hcy. For GSH, the cyclization step is kinetically
unfavorable as it would involve the formation of either 11- or
a.
H. Tong and J. Zhao contributed equally to this work.
Shanghai Key Laboratory of New Drug Design, Shanghai Key Laboratory of
Chemical Biology, School of Pharmacy, and State Key Laboratory of
Bioengineering Reactor, East China University of Science & Technology, 130
Meilong Road, Shanghai 200237, China.
b.
1
2-membered ring. So the reaction products stay as the thiol-
Michael adducts (Scheme S2, ESI†). In terms of fluorescence
sensing mechanism, for the maleimide group, it is a well-
established quenching group through photo-induced electron
c.
Department of Chemistry and Chemical Biology, University of New Mexico,
Albuquerque, NM 87131-0001, USA.
transfer (PET) mechanism due to its low-lying empty π* orbital
*
To whom correspondence should be addressed.
K. Lou, Email: kylou@ecust.edu.cn; W. Wang, E-mail: wwang@unm.edu.
Electronic Supplementary Information (ESI) available: Synthetic procedures, NMR,
30, 31
of the double bond.
The fast thiol-Michael addition removes
†
the PET effect, resulting in a quick and usually drastic turn-on
HRMS, and other UV-Vis and fluorescent spectra. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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fluorescence response.32 Since this step only involves thiol
Journal Name
Probe
1 was conveniently synthesized from 3-amino-7-
View Article Online
33
DOI: 10.1039/C6CC09336A
group, thus it is not selective for Cys, Hcy, and GSH. The slow hydroxycoumarin (
4
)
in two steps with high yield (Scheme S3,
transcyclization step may change fluorescence emission but ESI†). The coumarin
4
first reacted with maleic anhydride to
generally not as drastic as the thiol-Michael step. Therefore, form the maleic amide acid 5. Then the acid 5 was stirred with
maleimide-based fluorescent probes are generally not selective excess acryloyl chloride in the presence of triethylamine. The
among different thiols. In contrast, fluorescence sensing hydroxyl group was protected as an acrylate group, and the
mechanism of acrylate-based fluorescent probes are mainly maleic amide acid was simultaneously converted to
based on the change of internal charge transfer (ICT) at the maleimide group. When acyl chloride was used instead of
excited state due to the change of substituent from the acryloyl chloride, the reference probe was obtained (Scheme
a
6
electron-withdrawing ester to the electron-donating hydroxyl S5, ESI†). Similarly the reference probe
7
was synthesized from
group in the cyclization-deprotection step.23 Therefore, the 3-amino-7-methoxy-coumarin
(
8
)
34
(Scheme S6, ESI†).
1
acrylate based fluorescent probes were generally selective for Structures of these probes were firmly confirmed by HNMR,
1
3
Cys/Hcy over GSH (Scheme 1, Scheme S2, ESI†). Based on the
above reaction and sensing mechanisms, we envisioned that
CNMR, and HRMS (see ESI†).
With the probe in hand, we first tried model reaction of
with excess amount of cysteine methyl ester (Cys-OMe)
1
incorporation of the acrylate and maleimide groups onto a probe
1
fluorophore would result in a fluorescent probe adopting dual in a mixed solvent of DMSO and water. An inseparable mixture
cyclization processes to achieve increased kinetic differences of two diastereomers in a ratio about 3:2 were isolated based
1
for improved selectivity of Cys over Hcy. In order to fully on their HNMR spectrum. The double bond absorption peaks
translate reaction kinetic differences of dual cyclizations to of the acrylate (δ 6.60, 6.45, 6.23) and the maleimide group (δ
fluorescence signal readout for high selectivity, the electron- 7.32) were disappeared, indicating that both groups were
withdrawing acrylate and the maleimide group have to be put reacted (Fig. S13, ESI†). HPLC-MS studies of probe
1 with 40
at the two ends of a fluorophore for efficient ICT quenching and equiv. of Cys-OMe confirmed the removal of the acrylate group
allows drastic fluorescence turn-on response only after dual and addition of 1 equiv. of Cys-OMe in the final product.
cyclization processes (Scheme 1).
However, whether the transcyclization happened after thiol-
Michael addition of the maleimide group was difficult to
confirm. We then turned to the model reaction with
cysteamine. When probe 1 reacted with 1 equiv. of cysteamine
in THF for 5 min, compound 2a was isolated in 64% yield, in
which the acrylate group was retained while the maleimide
group was converted to 6-membered thiomorpholinone ring
through thiol-Michael addition and transcyclization cascade
reaction. The formation of 6-membered ring was confirmed by
2
D-NOESY spectrum (Fig. S18, ESI†). The compound 2a was used
as a reference probe only containing an acrylate group. When 2
equiv. of Cys were used, product 3a was isolated in 84% yield.
Similarly, its 6-membered ring was confirmed by 2D-NOESY
spectrum (Fig. S17, ESI†). The second equiv. of cysteamine
reacted with the acrylate group and deprotected it through
thiol-Michael addition and cyclization-deprotection cascade
sequence. The model reactions so far supported that both
reactive groups in probe
1 should follow established
mechanisms when reacted with 2-aminothiols, including
cysteamine, Cys-OMe, and Cys.
We then studied fluorescence response of probe
1 with Cys.
Initial fluorescence titration of probe (1 μM) towards
1
Scheme 1 Schematic illustration of synergetic PET and ICT effect of increasing amount of Cys showed a concentration-dependent
the maleimide and acrylate groups in a single fluorophore.
fluorescence turn-on response toward addition of Cys. A
minimum 40 equiv. Cys was required to achieve a maximum
fluorescence turn-on response in 30 min (Fig. S1, ESI†). Time-
dependent fluorescence emission spectra of the probe 1 (1 μM)
with 40 μM Cys verified maximum fluorescence response was
achieved in 30 min (Fig. 2c). The probe to Cys ratio of 40 and
incubation time of 30 min were selected for further
Fig. 1 Structure of fluorescent probe 1 and the reference fluorescent
probes
.
photophysical studies. Under this condition, the probe
and turn-on fluorescence at 476 nm with very low fluorescence
7 using 3-amino-7- background (Fig. 2a). The maximum fluorescence excitation and
1 gave
To test our hypothesis, we designed the target probe
three reference probes 2a , and
hydroxycoumarin ( ) as the model fluorophore (Fig. 1).
1
,
6
4
emission wavelength were 340 nm and 476 nm, respectively
2
| J. Name., 2012, 00, 1-3
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Journal Name
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(
1
Fig. S3, ESI†). The normalized fluorescence spectrum of probe were likely converted to the thiol-Michael adductVsiebwuAtrtincleoOt nylienet
incubation with 40 equiv. Cys for 30 min almost overlapped underwent cyclization (Fig. S8, ESI†). DTOhIe: 10s.t1r0u3c9t/uCr6eCCo0f93t3h6eA
with that of product 3a isolated from the model reaction of intermediate
probe
the reaction of probe
with cysteamine. The detection limit of probe
to be 13.7 nM at S/N=3 at low Cys concentrations (0 to 5 μM),
Fig. S5, ESI†).
2
and the proposed reaction mechanism was
e
1
with 2 equiv. of cysteamine (Fig. S4, ESI†), suggesting shown in Scheme S7 (see ESI†).
1
with Cys follows the same mechanism
was determined
a
1
(
0
.8
probe 1
probe 1 + Cys
1
200000
probe 1
probe 1 + Cys
b)0.7
a)
1
000000
0
.6
.5
8
6
4
2
00000
00000
00000
00000
0
0
0.4
0.3
b
f
g
h
0.2
0.1
0.0
3
50
400
450
500
550
600
650
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
0.8
30 min
d)0.7
1
000000
c)
0.6
0.5
0.4
4 min
800000
600000
400000
200000
0
30 min
4 min
c
0
0.3
2 min
min
30 min
0
0.2
0.1
0.0
350
400
450
500
550
600
650
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
Cys
1400000
1200000
f)
e)1000000
400 equiv. other amino
acids, metal ions, glucose,
H2O2, and blank
1200000
d
1
000000
8
6
4
2
00000
00000
00000
00000
0
800000
600000
400000
200000
0
Hcy
GSH
3
50
400
450
500
550
600
650
Wavelength (nm)
1
CysHcyGSH
2a CysHcyGSH
6
Cys HcyGSH
7 Cys HcyGSH
Fig. 3 Fluorescence images (e-h) collected at 430-495 nm (blue to
cyan-blue, λex = 400 nm) and the corresponding bright field view (a-
d) of HeLa cells after different treatment. a) and e) were pretreated
with 1 mM N-ethylmaleimide (NEM) before incubation with the
probe 1 for 1 h. (b, f), (c, g), and (d, h) were first pretreated with 1
mM NEM 1 h, then pretreated with 500 µM Cys, Hcy, and GSH for
Fig. 2 a) The fluorescence emission spectra of probe 1 (1 μM) in the
absence and presence of 40 µM Cys in PBS buffer. (λex = 340 nm,
incubation time = 30 min); b) Absorption spectra of probe 1 (50 µM)
in the absence and presence of 2 mM Cys in PBS buffer (incubation
time = 30 min); c) time-dependent fluorescence emission spectra of 1
μM probe 1 upon addition of 40 µM Cys in PBS buffer (λex = 340
nm, measured in every two minutes); d) time-dependent absorption
spectra of probe 1 (50 µM) upon addition of Cys (2 mM); e)
Fluorescence spectra of probe 1 (1 µM) (λex = 340 nm) toward various
species in PBS buffer (10 mM, pH 7.4) including 40 equiv.
aminothiols (Cys, Hcy, GSH), 400 equiv. different amino acids (Gly,
Lys, Arg, His, Tyr, AsP, Thr, Trp, Glu, Pro), 400 equiv. metal ions
3
0 min respectively, finally incubated with 20 µM probe 1 for 1 h
(scale bar = 100 µM).
We then investigated selectivity of the probe
1 for Cys
detection over Hcy, GSH, other amino acids, metal ions,
hydrogen peroxide, and glucose (Fig. 2e and Fig. S10, ESI†). For
+
2+
+
2+
2+
3+
various species tested so far, probe
1
showed good to excellent
(
2 2
K , Ca , Na , Mg , Zn , Fe ), 400 equiv. H O , and 400 equiv.
glucose. The control group was the probe 1 (1 µM) alone; f) selectivity for Cys. Notably, probe
1
showed enhanced
Comparision of fluorescence intensity at 476 nm (λex = 340 nm) of 1 selectivity for Cys than both the reference probe 2a and
7 (Fig.
µM probe 1, reference probe 2a, 6, and 7 toward 40 equiv. of Cys,
2
f) with only one recognition group. The ratios of fluorescence
Hcy, and GSH. (All measurements were taken in 10 mM PBS buffer
solution at pH = 7.4)
intensity at 476 nm of the fluorescent probe in response to Cys
versus to Hcy were increased from 3.7, and 1.6 for the probe 2a
More reaction details was revealed from UV-vis studies. and
Probe
showed two maximum absorption bands at 293 and gave much lowered fluorescence background and a drastic
18 nm. After reaction with 40 equiv. Cys for 30 min, the fluorescence turn-on (> 930-fold) response for Cys, while the
maximum absorption band red-shifted to 340 nm (Fig. 2b). fluorescence turn-on ratio was only 5.9-fold for the reference
Time-dependent absorption spectra of probe
clearly showed probe 2a. Compared with the probe , the reference probe
an isobestic point at about 370 nm after 4 min, indicating the had similarly low fluorescence background from dual PET and
formation of an intermediate (Fig. 2d). From comparison with ICT quenching mechanisms, but gave much lowered
the time-dependent UV-vis spectra of probe
with 40 equiv. N- fluorescence turn-on response, indicating the importance of the
7, respectively, to 5.5 for the probe 1. Moreover, probe 1
1
3
1
1
6
a
1
acetyl cysteine (NAC) (Fig. S6, ESI†) and the reference probe 2a removal of acrylate group and associated ICT quenching effect
with 40 equiv. of NAC at extended time (Fig. S7, ESI†), it was for the observed drastic fluorescence turn-on response of probe
deduced at the time point of 4 min, both the recognition groups
1
. The dual quenching mechanisms and drastic fluorescence
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Journal Name
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B. D'Agostino, P. W. F. Wilson and P. A. WDOolIf:,1N0.e10w39E/nCg6l.CCJ.09M3e36dA.,
002, 346, 476-483.
. A. A. Mangoni and S. H. D. Jackson, Am. J. Med., 2002, 112, 556-
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supported by the measured fluorescence quantum yields of the
probe , reference probe 2a, and product 3a as 0.003, 0.149,
and 0.809, respectively (Fig. S9, ESI†).
The selectivity of probe for detection of Cys over Hcy and
GSH was further investigated by cell imaging studies. Before cell
studies, pH-dependent studies (Fig. S11, ESI†) showed probe
was suitable for use at biological pHs from 4-8, while MTS assay
demonstrated that the probe did not have significant toxicity
up to 100 µM to HeLa cells (Fig. S14). Then, HeLa cells were
2
1
9
1
1
5
1
0. D. M. Townsend, K. D. Tew and H. Tapiero, Biomed.
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30, 10846-10847.
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to increase the specific thiol level inside cells, and further
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1 (20 μM in PBS containing 0.2% DMSO)
at 37 °C for 1 h before cells were imaged using a fluorescence
microscope at the blue channel (430-495 nm). As the control
group, cells with only NEM treatement and incubation with
2
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pretreated with NEM (1 mM) gave almost no blue fluorescence
(
Fig. 3e), while Cys-incubated cells (Fig. 3f) exhibits strong blue 18. Y. Tang, D. Lee, J. Wang, G. Li, J. Yu, W. Lin and J. Yoon, Chem.
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3
4
above cell imaging studies clearly demonstrated that probe 1 is
capable of selective detection and imaging of Cys over Hcy and
GSH in cells.
In summary, we presented here a new approach to achieve
enhanced selectivity for detection of Cys over Hcy and GSH
through dual cyclization processes and dual PET and ICT
quenching mchanism. This approach was demonstrated by
incorporation of an acrylate and a maleimide group onto two
opposite sides of a single coumarin fluorophore, 3-amino-7-
2
2
2
0. L.-Y. Niu, Y.-Z. Chen, H.-R. Zheng, L.-Z. Wu, C.-H. Tung and Q.-Z.
Yang, Chem. Soc. Rev., 2015, 44, 6143-6160.
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drastically improved fluorescence turn-on response and
enhanced selectivity for detection of Cys over Hcy and GSH than
using either the acrylate or the maleimide group alone. We also
demonstrated the capability of the probe for selective turn-on
fluorescence detection of Cys over Hcy and GSH in cell imagings.
The work was supported by East China University of Science
and Technology (start-up funds, W. W.), the Fundamental
Research Funds for the Central Universities (K. L., WY1213013),
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