Thermo-triggered release of a Cys probe from the cavity of a water-soluble pillar[5]arene

Article abstract of DOI:10.1016/j.tetlet.2015.06.005

Abstract A novel [2]pseudorotaxane based on thermo-responsive water-soluble pillar[5]arene WP5 and Cys probe 1 was successfully prepared in water. This complex exhibited no responsiveness to Cys at room temperature due to its high stability that inhibited

Full text of DOI:10.1016/j.tetlet.2015.06.005

Tetrahedron Letters 56 (2015) 4545–4548
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Thermo-triggered release of a Cys probe from the cavity of a water-
soluble pillar[5]arene
⇑
Xiaodong Chi, Pi Wang, Yang Li, Xiaofan Ji
Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel [2]pseudorotaxane based on thermo-responsive water-soluble pillar[5]arene WP5 and Cys probe
1 was successfully prepared in water. This complex exhibited no responsiveness to Cys at room temper-
ature due to its high stability that inhibited the interaction between probe 1 and Cys. However, the
encapsulated 1 could be released from the cavity of WP5 upon heating, leading to the formation of
1–Cys adduct. Moreover, the probe could be used to monitor Cys with high sensitivity. Importantly,
WP5, 1, and the [2]pseudorotaxane WP5ꢀ1 showed excellent biocompatibility, making the probe a very
attractive candidate for biological applications.
Received 23 April 2015
Revised 1 June 2015
Accepted 3 June 2015
Available online 6 June 2015
Keywords:
Pillararene
Hydrophobic interaction
Host–guest system
Supramolecular chemistry
Ó 2015 Elsevier Ltd. All rights reserved.
Over the past few years, the development of probes for thiols
has been an active research area because of the biological impor-
tance of thiols.1 Thiol-containing small molecules, such as cysteine
(Cys), homocysteine (Hcy), and glutathione (GSH), play crucial
roles in living organisms, and are involved in a number of biologi-
cal processes and significantly associated with a wide variety of
diseases.2 In view of the biological importance of thiols, it is not
surprising that there has been an increasing interest in the design
of analytical methodologies for their detection. Recently, consider-
able efforts have been made to develop probes with specific prop-
erties and functions revealing their affinity and selectivity toward
certain thiols. However, due to the instant interaction between thi-
ols and their corresponding probes as soon as they met each other,
it is too difficult for the probes to detect thiols in the targeted areas.
Moreover, many of these strategies may also bring about some
unexpected problems, such as cytotoxicity and low selectivity,
confining their applications in biology. Therefore, it is still a big
challenge to develop thiol-specific probe systems with biocompat-
ibility to detect thiols in specific regions as we expected.3
porous materials.10 Especially, neutral water-soluble pillar[n]are-
nes bearing triethylene oxide chains have been demonstrated to
be excellent hosts for molecules of various sizes and shapes in
water.11 Moreover, these water-soluble pillar[n]arenes show lower
critical solution temperature (LCST) behavior, which can undergo
phase separation when the temperature exceeds the LCST, disas-
sembling the pillar[n]arene-based host–guest complexes.
Herein we report that a water-soluble pillar[5]arene bearing
triethylene oxide chains WP5 (Scheme 1) that can interact with a
new probe 1, which can selectively monitor Cys among common
thiol-containing small molecules, to form an inclusion complex
in water, inhibiting the interaction between probe 1 and Cys.
Due to the thermo-responsiveness of the WP5 host, the encapsuled
probe 1 can be released from the cavity of WP5 when the temper-
ature exceeds the LCST, leading to the formation of 1–Cys adduct.
Moreover, WP5, probe 1 and WP5ꢀ1 showed excellent biocompat-
ibility, making the probe a very attractive candidate for biological
applications.
The host–guest complexation between WP5 and 1 was firstly
studied by 1H NMR experiments (Fig. 1). Compared with the spec-
trum of free 1 (Fig. 1a), upfield shift changes were observed for the
Pillar[n]arenes,4,5 a new generation of macrocyclic hosts next to
crown ethers,6 cyclodextrins,7 calixarenes,8 and cucurbiturils,9
have been developed rapidly because of their unique structures
and high functionality, bringing along many applications in the
fabrication of interesting and functional supramolecular systems,
such as supramolecular polymers, drug delivery systems, trans-
membrane channels, thermo-responsive materials, and hybrid
signals related to the protons on 1 (
D
d = À0.75, À2.02 and À1.35
for H2, H4, and H10, respectively) upon the addition of a molar
equivalent amount of WP5 (Fig. 1b). Moreover, the peaks of pro-
tons on WP5 also exhibited slight chemical shift changes in the
presence of 1 arising from the interactions between WP5 and 1
(Fig. 1b). Furthermore, some signals related to the protons on 1 dis-
appeared when WP5 was mixed with a molar equivalent of guest 1
in water. This may attribute to the formation of the inclusion
⇑
Corresponding author.
E-mail address: xiaofanji@zju.edu.cn (X. Ji).
http://dx.doi.org/10.1016/j.tetlet.2015.06.005
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

4546
X. Chi et al. / Tetrahedron Letters 56 (2015) 4545–4548
Scheme 1. Chemical structures of WP5, 1, and Cys and a cartoon representation of thermo-triggered release of Cys probe 1 from the cavity of WP5.
When an aqueous solution of 1 was mixed with 3 equiv of Cys,
the light red solution changed to colorless, suggesting the forma-
H10
H9
tion of 1–Cys adduct (Fig. 2a). UV–vis spectroscopy characteriza-
tion of the interaction between Cys and 1 was carried out at
room temperature in water (Fig. 2b). As the content of Cys
increased, the absorption peak at about 450 nm decreased gradu-
ally, and a new absorption peak appeared at 350 nm at the same
time. A well-defined isosbestic point was noted at 388 nm, which
indicated the formation of a new kind of species.2a The hyp-
sochromic shift in absorption was observed, implying that the
intramolecular charge transfer (ICT) from the electron-rich
dimethylamino moiety to the electron-poor pyridinium moiety
was turned off in the adduct.1 In addition, the formation of the
1–Cys adduct was also confirmed by mass spectrometric analysis
(Fig. S9, SI). What is more interesting is that probe 1 could monitor
Cys with high sensitivity, but not GSH, and shows low sensitivity to
Hcy. It was seen that the absorbance intensity at 350 nm of 1
induced by Cys increased much faster than that by Hcy (Fig. 2c),
H7
H3
H5
H8
H1H2
H4
H6
(a)
(b)
(c)
implying that
1 showed more sensitivity to Cys than Hcy.
Additionally, when excess GSH was added to the aqueous solution
of WP5, the absorption peak at 450 nm decreased slightly and no
new peaks appeared (Fig. 2c). These results indicated that 1 could
selectively monitor Cys.
Figure 1. Partial 1H NMR spectra (400 MHz, D2O, 298 K): (a) 2.00 mM 1; (b)
2.00 mM 1 and WP5; (c) 2.00 mM WP5.
We then studied whether the inclusion complex could effect the
responsiveness of 1–Cys by using the UV–vis method (Fig. 2d). It
can be seen that the inclusion complex exhibited almost no
response with the addition of Cys. Owing to the cavity of WP5,
probe 1 stayed in the cavity of WP5 and Cys could not meet 1 so
that it showed no responsiveness to Cys. Therefore, we conclude
that the inclusion complex effected the responsiveness of 1–Cys.
It was seen that the absorbance intensity of 1 induced by 3 equiv
of Cys increased much faster (Fig. 2b). However, for WP5ꢀ1, with
the presence of 3 equiv or even 6 equiv of Cys, it exhibited almost
no change in about 10 min (Fig. 2d). These results indicated that
WP5 played a crucial role in the inhibition for the interaction of
1 with Cys.
complex between WP5 and 1. When the inclusion complex
formed, the protons of 1 were located in the shielding region of
the cyclic pillar structure, leading to the disappearance of protons
H1, H3, H5, H6, H7, and H8.
To establish the binding mode between WP5 and 1, a 2D NOESY
experiment was carried out. The NOESY spectrum (Fig. S7, SI) of an
equimolar solution of WP5 and 1 shows cross peaks A, B, C, and D,
representing the correlations between the signals of protons H2
and H4 of 1 and those of the protons on the glycol chains of
WP5. All these results indicated that the complexation of WP5
with 1 took place in solution. Thus, we concluded that when 1
was mixed with WP5, it penetrated through the cavity of WP5
and stayed in it.
In a previous work,12 Ogoshi et al. reported that WP5 showed
LCST behavior. Hence, we hypothesized that the assembly and dis-
assembly of the WP5ꢀ1 complex can be reversibly controlled by
heating and cooling. As the temperature of the aqueous solution
increased to 60 °C, the signals of protons H1, H3, and H5–7 appeared
at their uncomplexed positions (Figs. S10 and S11a and b, SI).
The stoichiometry of the complex between WP5 and 1 was
determined to be 1:1 in aqueous solution by isothermal titration
calorimetry (ITC) and the association constant for the complex
was 4.21 ( 0.34) Â 104 MÀ1 (Fig. S8, SI).

X. Chi et al. / Tetrahedron Letters 56 (2015) 4545–4548
4547
1.5
(a)
(c)
(b)
1.0
0.5
0.0
350
400
450
500
550
Wavelength (nm)
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
a
b
1 alone
1 + WP5
(d)
+ 3 equiv Cys
1 + WP5
1 + WP5
d
1alone
1+Cys
1+GSH
1+Hcy
a
b
+ 6 equiv Cys
a
b
c
c
d
d
300 350 400 450 500 550
350 400 450 500 550
Wavelength (nm)
Wavelength (nm)
Figure 2. (a) Color change photographs for 1 (0.100 mM, left) and 1 + Cys (0.100 mM, right). (b) UV–vis spectra of the aqueous solution of 1 (0.100 mM) upon the addition of
various concentrations of Cys (0.00–3.00 equiv). (c) UV–vis spectra of aqueous solution of 1 with different thiols. (d) Absorption intensity of 1 (0.100 mM) upon the addition of
WP5 and Cys in water.
However, as the temperature of this solution cooled down to 25 °C,
the complexation between WP5 and 1 was recovered and the sig-
nals of protons H1, H3, and H5–7 of 1 disappeared again (Fig. S11c,
SI). Therefore, we demonstrated that the assembly and disassem-
bly of the WP5ꢀ1 complex could be successfully controlled by
heating or cooling, providing a convenient switch to fabricate novel
responsive supramolecular materials.
Furthermore, we tested the responsiveness of 1–Cys after
released from the cavity of WP5. As shown in Figure 3, the absorp-
tion peaks at 450 nm of the UV–vis curve of 1 + WP5 at 60 °C were
higher than those of 1 + WP5 at 25 °C, proving the thermo-respon-
siveness of the WP5ꢀ1 complex. Moreover, the absorption peaks at
450 nm decreased and a new peak appeared at 350 nm upon the
subsequent addition of 3 equiv of Cys at 60 °C, implying that the
interaction between 1 and Cys recovered after the removal of
WP5. This thermo-responsive rapid release of 1 can be explained
by the introduction of a thermo-triggered inclusion complex, mak-
ing it an ideal candidate for probe release.
evaluate the potential toxicity of the supramolecular assemblies.
Figure 4 shows the relative cell viability of the Hela cells deter-
mined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay after incubating with various concentrations
of carriers for 24 h. As shown in Figure 4, relative cell viability of
Hela cells cultured with WP5ꢀ1, WP5 and 1 showed minimal
influence on cell viability at all detected concentrations. These data
provided convincing support that biologically relevant applications
of WP5 can be achieved in the future. We also believe that the good
performance on biocompatibility will make the probe very attrac-
tive for practical applications.
In summary, we have successfully constructed a novel inclusion
complex between thermo-responsive water-soluble pillar[5]arene
WP5 and Cys probe 1 in water. This complex exhibited no respon-
siveness to Cys at room temperature due to the formation of the
stable inclusion complex that inhibited the interaction between
probe 1 and Cys. The encapsulated 1 could be released from the
It is well known that biocompatibility is a basic requirement for
an ideal biological detection system. Thus, we selected Hela cells to
WP5
120
90
60
30
0
1
WP5+1
a
1 alone
1 + WP5 at 60 ºC
1 + WP5 at 25 ºC
1.5
1.0
0.5
0.0
b
c
1 + WP5 + 3 equiv Cys at 60 ºC
d
a
b
c
d
350
400
450
500
550
30
60
90
120
150
Wavelength (nm)
Concentration
Figure 3. Absorption spectra: (a) 0.200 mM 1; (b) 0.200 mM 1 and WP5 at 60 °C;
(c) 0.200 mM 1 and WP5 at 25 °C; (d) addition of 3 equiv Cys to (b).
Figure 4. Cytotoxicity of 1, WP5, and the host–guest complex WP5ꢀ1 against Hela
cells.

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X. Chi et al. / Tetrahedron Letters 56 (2015) 4545–4548
Commun. 2014, 852; (h) Strutt, N. L.; Zhang, H.; Stoddart, J. F. Chem. Commun.
2014, 7455; (i) Li, Z.-Y.; Zhang, Y.; Zhang, C.-W.; Chen, L.-J.; Wang, C.; Tan, H.;
Yu, Y.; Li, X.; Yang, H.-B. J. Am. Chem. Soc. 2014, 136, 8577; (j) Zhou, J.; Chen, M.;
Diao, G. Chem. Commun. 2014, 11954.
cavity of WP5 upon heating, recovering the formation of 1–Cys
adduct. Moreover, WP5, probe 1, and WP5ꢀ1 showed good bio-
compatibility. Therefore, thermo-responsive and biocompatible
supramolecular inclusion complex WP5ꢀ1 has potential applica-
tions in the quantitative detection of Cys, and will provide an
important pathway for guiding the design of functional
supramolecular biomaterials.
5. (a) Cao, D.; Kou, Y.; Liang, J.; Chen, Z.; Wang, L.; Meier, H. Angew. Chem., Int. Ed.
2009, 48, 9721; (b) Guan, Y.; Ni, M.; Hu, X.; Xiao, T.; Xiong, S.; Lin, C.; Wang, L.
Chem. Commun. 2012, 8529; (c) Yu, G.; Zhou, X.; Zhang, Z.; Han, C.; Mao, Z.;
Gao, C.; Huang, F. J. Am. Chem. Soc. 2012, 134, 19489; (d) Hu, X.-B.; Chen, Z.;
Chen, L.; Zhang, L.; Hou, J.-L. Chem. Commun. 2012, 10999; (e) Xu, J.-F.; Chen, Y.-
Z.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. Org. Lett. 2013, 15, 6148; (f) Li, H.; Chen,
D.-X.; Sun, Y.-L.; Zheng, Y.; Tan, L.-L.; Weiss, P. S.; Yang, Y.-W. J. Am. Chem. Soc.
2013, 135, 1570; (g) Chi, X.; Ji, X.; Xia, D.; Huang, F. J. Am. Chem. Soc. 2015, 137,
1440.
Acknowledgment
6. (a) Niu, Z.; Gibson, H. W. Chem. Rev. 2009, 109, 6024; (b) Niu, Z.; Huang, F.;
Gibson, H. W. J. Am. Chem. Soc. 2011, 133, 2836; (c) Zhu, K.; Vukotic, V. N.; Loeb,
S. J. Angew. Chem., Int. Ed. 2012, 51, 2168; (d) Qi, Z.; Molina, P. M.; Jiang, W.;
Wang, Q.; Nowosinski, K.; Schulz, A.; Gradzielski, M.; Schalley, C. A. Chem. Sci.
2012, 3, 2073; (e) Chen, L.; Tian, Y.; Ding, Y.; Tian, Y.; Wang, F. Macromolecules
2012, 45, 8412; (f) Yan, X.; Xu, D.; Chi, X.; Chen, J.; Dong, S.; Ding, X.; Yu, Y.;
Huang, F. Adv. Mater. 2012, 24, 362; (g) Tian, Y.; Chen, L.; Tian, X.-Y.; Wang, X.;
Wang, F. Polym. Chem. 2013, 4, 453.
7. Ma, X.; Tian, H. Acc. Chem. Res. 2014, 47, 1971.
8. Guo, D.-S.; Liu, Y. Chem. Soc. Rev. 2012, 41, 5907.
9. (a) Isaacs, L. Acc. Chem. Res. 2014, 47, 2052; (b) Yang, H.; Ma, Z.; Wang, Z.;
Zhang, X. Polym. Chem. 2014, 5, 1471; (c) Yang, H.; Yuan, B.; Zhang, X.;
Scherman, O. A. Acc. Chem. Res. 2014, 47, 2106.
10. (a) Zhang, Z.; Luo, Y.; Chen, J.; Dong, S.; Yu, Y.; Ma, Z.; Huang, F. Angew. Chem.,
Int. Ed. 2011, 50, 1397; (b) Duan, Q.; Xia, W.; Hu, X.; Ni, M.; Jiang, J.; Lin, C.; Pan,
Y.; Wang, L. Chem. Commun. 2012, 8532; (c) Hu, X.-B.; Chen, Z.; Tang, G.; Hou,
J.-L.; Li, Z.-T. J. Am. Chem. Soc. 2012, 134, 8384; (d) Wang, X.; Han, K.; Li, J.; Jia,
X.; Li, C. Polym. Chem. 2013, 4, 3998; (e) Hu, X.-Y.; Wu, X.; Wang, S.; Chen, D.;
Xia, W.; Lin, C.; Pan, Y.; Wang, L. Polym. Chem. 2013, 4, 4292; (f) Dong, S.;
Zheng, B.; Yao, Y.; Han, C.; Yuan, J.; Antonietti, M.; Huang, F. Adv. Mater. 2013,
25, 6864; (g) Li, C. Chem. Commun. 2014, 12420.
11. (a) Ogoshi, T.; Shiga, R.; Yamagishi, T.-A. J. Am. Chem. Soc. 2012, 134, 4577; (b)
Chen, H.; Fan, J.; Hu, X.; Ma, J.; Wang, S.; Li, J.; Yu, Y.; Jia, X.; Li, C. Chem. Sci.
2015, 6, 197.
12. (a) Ogoshi, T.; Kita, K.; Yamagishi, T.-A. J. Am. Chem. Soc. 2012, 134, 20146; (b)
Chi, X.; Xue, M. Chem. Commun. 2014, 13754.
This work was supported by the Fundamental Research Funds
for the Central Universities.
Supplementary data
Supplementary data (1H NMR, 2D NOESY spectra and ESI mass
spectra) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.tetlet.2015.06.005.
References and notes
1. Lee, M. H.; Yang, Z.; Lim, C. W.; Lee, Y. H.; Kang, S. Chem. Rev. 2013, 113, 5071.
2. Yang, X.-F.; Huang, Q.; Zhong, Y.; Li, Z.; Li, H.; Lowry, M.; Escobedo, J. O.;
Strongin, R. M. Chem. Sci. 2014, 5, 3183.
3. Kunzmann, M. H.; Bach, N. C.; Bauer, B.; Sieber, S. A. Chem. Sci. 2014, 5, 1158.
4. (a) Ogoshi, T.; Kanai, S.; Fujinami, S.; Yamagishi, T.; Nakamoto, Y. J. Am. Chem.
Soc. 2008, 130, 5022; (b) Zhang, Z.; Xia, B.; Han, C.; Yu, Y.; Huang, F. Org. Lett.
2010, 12, 3285; (c) Holler, M.; Allenbach, N.; Sonet, J.; Nierengarten, J.-F. Chem.
Commun. 2012, 2576; (d) Li, C.; Han, K.; Li, J.; Zhang, H.; Ma, J.; Shu, X.; Chen, Z.;
Weng, L.; Jia, X. Org. Lett. 2012, 14, 42; (e) Yu, G.; Han, C.; Zhang, Z.; Chen, J.;
Yan, X.; Zheng, B.; Liu, S.; Huang, F. J. Am. Chem. Soc. 2012, 134, 8711; (f) Yao, Y.;
Xue, M.; Chen, J.; Zhang, M.; Huang, F. J. Am. Chem. Soc. 2012, 134, 15712; (g)
Kothur, R. R.; Hall, J.; Patel, B. A.; Leong, C. L.; Boutelle, M. G.; Cragg, P. J. Chem.