Chromogenic and fluorogenic sensing of biological thiols in aqueous solutions using BODIPY-based reagents

Article abstract of DOI:10.1021/ol303306s

Judicious design of BODIPY dyes carrying nitroethenyl substituents in conjugation with the BODIPY core yields dyes that respond to biological thiols by both absorbance and emission changes. Incorporation of solubilizing ethyleneglycol units ensures water

Full text of DOI:10.1021/ol303306s

ORGANIC
LETTERS
2013
Vol. 15, No. 1
216–219
Chromogenic and Fluorogenic Sensing
of Biological Thiols in Aqueous Solutions
Using BODIPY-Based Reagents
Murat Isik,‡ Tugba Ozdemir,‡ Ilke Simsek Turan,‡ Safacan Kolemen,‡ and
Engin U. Akkaya*,†,‡
Department of Chemistry and UNAM-National Nanotechnology Research Center,
Bilkent University, 06800 Ankara, Turkey
eua@fen.bilkent.edu.tr
Received December 2, 2012
ABSTRACT
Judicious design of BODIPY dyes carrying nitroethenyl substituents in conjugation with the BODIPY core yields dyes that respond to biological
thiols by both absorbance and emission changes. Incorporation of solubilizing ethyleneglycol units ensures water solubility. The result is bright
signaling of biologically relevant thiols in the longer wavelength region of the visible spectrum and in aqueous solutions.
Sensing and signaling of biological thiols is at the focus
of recent flurry of work.1 These reaction-based probes
take advantage of high nucleophilicity of thiol functions
found in biologically relevant species such as Cystein
(Cys), Homocystein (Hcy) and Glutathione (GSH). These
biothiols are vital for the maintenance of cellular redox
status and alterations in their levels is linked toa number of
debilitating diseases such as AIDS, cancer and Alzheimer’s.2
Consequently, probes that respond to these thiols by color
change, emission change, or both are highly valued. If they
are made to function in aqueous solutions, naturally, they
are deemed to be more desirable considering potential
applications.
responses for fast, selective and sensitive detection of bio-
logical thiols in aqueous media. The roles of three separate
modules of the thiol probe 1 are displayed in Scheme 1 and
can be described as follows: (1) Boron dipyrromethene
(BODIPY)3 fluorophore was chosen as the signaling unit
for its widely accepted superiority (e.g., high molar ab-
sorptivity, high photostability, visible wavelength absorp-
tion and its modular nature for facile functionalization,
etc.) over more conventional alternatives. Because of these
unique features, a plethora of fluorescent sensors/labels,4
(3) (a) Ziessel, R.; Ulrich, G.; Harriman, A. New. J. Chem. 2007, 31,
496. (b) Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891. (c) Ulrich,
G.; Ziessel, R.; Harriman, A. Angew. Chem., Int. Ed. 2008, 47, 1184.
(4) (a) Sunahara, H.; Urano, Y.; Kojima, H.; Nagano, T. J. Am.
Chem. Soc. 2007, 129, 5597. (b) Baruah, M.; Qin, W. W.; Vallee, R.;
Beljonne, D.; Rohand, T.; Dehaen, W.; Boens, N. Org. Lett. 2005, 7,
4377. (c) Yin, S. C.; Leen, V.; Van Snick, S.; Boens, N.; Dehaen, W.
Chem. Commun. 2010, 46, 6329. (d) Qi, X.; Kim, S. K.; Han, S. J.; Xu, L.;
Jee, A. Y.; Kim, H. N.; Lee, C.; Kim, Y.; Lee, M.; Kim, S. J.; Yoon, J.
Tetrahedron Lett. 2008, 49, 261. (e) Niu, S.; Massif, C.; Ulrich, G.;
Renard, P.; Romieu, A.; Ziessel, R. Chem.;Eur. J. 2012, 18, 7229. (f)
Rurack, K.; Kollmansberger, M.; Resch-Genger, U.; Daub, J. J. Am.
Chem. Soc. 2000, 122, 968. (g) Bozdemir, O. A.; Guliyev, R.; Buyukcakir,
O.; Selcuk, S.; Kolemen, S.; Gulseren, G.; Nalbantoglu, T.; Boyaci, H.;
Akkaya, E. U. J. Am. Chem. Soc. 2010, 132, 8029. (h) Atilgan, S.;
Ozdemir, T.; Akkaya, E. U. Org. Lett. 2010, 47, 4792.
We now present a nitroethenyl-BODIPY conjugate
1, having both chromogenic and fluorescence turn-on
† Department of Chemistry
‡ UNAM-National Nanotechnology Research Center
(1) (a) Chen, X.; Zhou, Y.; Peng, X.; Yoon, J. Chem. Soc. Rev. 2010,
39, 2120. (b) Zhou, Y.; Yoon, J. Chem. Soc. Rev. 2012, 41, 51. (c) Jun,
M. E.; Roy, B.; Ahn, K. H. Chem. Commun. 2011, 47, 7583.
(2) (a) Zhang, S. Y.; Ong, C.-N.; Shen, H.-M. Cancer Lett. 2004, 208,
143. (b) Pastore, A.; Piemonte, F.; Locatelli, M.; Lo Russo, A.; Gaeta,
L. M.; Tozzi, G.; Federici, G. Clin. Chem. 2001, 47, 1467. (c) Townsend,
D. M.; Tew, K. D.; Tapiero, H. Biomed. Pharmacother. 2003, 57, 145.
r
10.1021/ol303306s
Published on Web 12/20/2012
2012 American Chemical Society

light harvesting systems,5 photodynamic therapy agents6
and molecular logic gate systems7 are built around BOD-
IPY cores, and these received considerable attention from
the chemical community. (2) Incorporation of a nitroalk-
ene unit to the parent dye would transform it into a strong
Michael acceptor, which would be highly susceptible to
sulfhydryl nucleophiles.8 Indeed, Michael reaction-based
thiol sensing protocols9 clearly emerge as the favorite
among other strategies.10 Nucleophilic attack of the thiol
to the β-position of nitroethene is expected to disrupt the
π-conjugation and block the intramolecular charge trans-
fer (ICT) process, which is expected toinduce a blue shift in
absorbance. (3) Gallic acid derived unit placed at the meso-
position of BODIPY core plays two crucial functions:
(i) photoinduced-electron-transfer (PeT)-based modula-
tion of the emission, as the electron rich trimethoxyphenyl
moiety could quench excited state of the electron deficient
(due to conjugated nitroethenyl group) BODIPY core by
an electron transfer and (ii) ethyleneglycolic entities an-
chored on phenolic hydroxyl functionalities should facil-
itate water solubility, enhancing the chances for practical
applications of the probe 1.
Scheme 1. Design Elements of the Nitroethenyl-BODIPY
Conjugate Thiol Probe 1
(5) (a) Zhang, X.; Xiao, Y.; Qian, X. Org. Lett. 2008, 10, 29. (b) Iehl,
J.; Nierengarten, J.-F.; Harriman, A.; Bura, T.; Ziessel, R. J. Am. Chem.
Soc. 2012, 143, 988. (c) Bozdemir, O. A.; Cakmak, Y.; Sozmen, F.;
Ozdemir, T.; Siemiarczuk, A.; Akkaya, E. U. Chem.;Eur. J. 2010, 16,
6346. (d) Bozdemir, O. A.; Erbas-Cakmak, S.; Ekiz, O. O.; Dana, A.;
Akkaya, E. U. Angew. Chem., Int. Ed. 2011, 50, 10907.
(6) (a) Kamkaew, A.; Lim, S. H.; Lee, H. B.; Kiew, L. V.; Chung,
L. Y.; Burgess, K. Chem. Soc. Rev. 2013, 42, 77. (b) Yogo, T.; Urano, Y.;
Ishitsuka, F.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 12162. (c)
Gallagher, W. M.; Allen, L. T.; O’Shea, C.; Kenna, T.; Hall, M.;
Gorman, A.; Killoran, J.; O’Shea, D. F. J. Cancer 2005, 92, 1702. (d)
Erbas, S.; Gorgulu, A.; Kocakusakogullari, M.; Akkaya, E. U. Chem.
Commun. 2009, 4956. (e) Cakmak, Y.; Kolemen, S.; Duman, S.; Dede,
Y.; Dolen, Y.; Kilic, B.; Kostereli, Z.; Yildirim, L. T.; Dogan, A. L.;
Guc, D.; Akkaya, E. U. Angew. Chem., Int. Ed. 2011, 50, 11937.
(7) (a) Guliyev, R.; Ozturk, S.; Kostereli, Z.; Akkaya, E. U. Angew.
Chem., Int. Ed. 2011, 50, 9826. (b) Coskun, A.; Deniz, E.; Akkaya, E. U.
Org. Lett. 2005, 7, 5187.
To test the aforementioned hypothesis, we set out to
synthesize a simpler analogue of 1 and studied its 1,4-
addition reactivity with thiols. Thiol sensor 2 was prepared
from the readily available 2-formylBODIPY6e precursor
via tandem Henry/elimination reaction (see Supporting
Information). Once β-mercaptoethanol (ME), chosen as
thesimplebiothiol modelcompound, reacted withthe thiol
sensor 2, an apparent color change from red to orange was
noticed. This bright green fluorescing adduct 3 was pre-
sumed to be the 1,4-conjugate addition product (Figure 1).
Because of poor solubility of 2 in common polar organic
solvents, absorption and emission spectra were not re-
corded. Michael reaction of 2 and ME was amenable to
1H NMR spectroscopy analysis. Comparison of 1H NMR
spectra of 2 and 3 clearly shows that the addition of ME
to the sensor 2 leads to the disappearance of the vinylic
protons (Ha and Hb) resonating at 8.07 and 7.44 ppm with
the concomitant appearance of R-protons of the newly
formed nitroalkane showing at 4.80 ppm. About 0.1 ppm
upfield shift of aromatic hydrogens (meso-H and 6-H)
(8) Two nitroolefin-attached fluorescent thiol sensors were published
during the preparation of this manuscript. See: (a) Sun, Y.-Q.; Chen, M.;
Liu, J.; Lv, X.; Li, J.-f.; Guo, W. Chem. Commun. 2011, 47, 11029. (b)
Zhang, M.; Wu, Y.; Zhang, S.; Zhu, H.; Wu, Q.; Jiao, L.; Hao, E. Chem.
Commun. 2012, 48, 8925.
(9) (a) Yang, X.; Guo, Y.; Strongin, R. M. Angew. Chem., Int. Ed.
2011, 50, 10690. (b) Lim, S.; Escobedo, J. O.; Lowry, M.; Xu, X.;
Strongin, R. Chem. Commun. 2010, 46, 5707. (c) Ros-lis, J. V.; Garcia,
B.; Jimenez, D.; Martinez-Manez, R.; Sancenon, F.; Soto, J.; Gonzalvo,
F.; Valldecabres, M. C. J. Am. Chem. Soc. 2004, 126, 4064. (d) Hong, V.;
Kislukhin, A. A.; Finn, M. G. J. Am. Chem. Soc. 2009, 131, 9986. (e)
Huo, F. J.; Sun, Y.-Q.; Su, J.; Chao, J. B.; Zhi, H. J.; Yin, C. X. Org. Lett.
2009, 11, 4918. (f) Jung, H. S.; Ko, K. C.; Kim, G.; Lee, A.; Na, Y.;
Kang, C.; Lee, J. Y.; Kim, J. S. Org. Lett. 2011, 13, 1498. (g) Yuan, L.;
Lin, W.; Yang, Y. Chem. Commun. 2011, 47, 6275. (h) Lin, W.; Yuan, L.;
Cao, Z.; Feng, Y.; Long, L. Chem.;Eur. J. 2009, 15, 5096. (i) Chen, X.;
Ko, S.; Kim, M. J.; Shin, I.; Yoon, J. Chem. Commun. 2010, 46, 2751. (j)
Lee, J.; Lee, S.; Zhai, D.; Ahn, Y.; Yeo, H. Y.; Tan, Y. L.; Chang, Y.
Chem. Commun. 2011, 47, 4508. (k) Sun, Y.; Chen, M.; Liu, J.; Lv, X.; Li,
J.; Guo, W. Chem. Commun. 2011, 47, 11029. (l) Kwon, H.; Lee, K.;
Kim, H. Chem. Commun. 2011, 47, 1773. (m) Kim, G.; Lee, K.; Kwon,
H.; Kim, H. Org. Lett. 2011, 13, 2799.
(10) (a) Rusin, O., St.; Luce, N. N.; Agbaria, R. A.; Escobedo, J. O.;
Jiang, S.; Warner, I. M.; Dawan, F. B.; Lian, K.; Strongin, R. M. J. Am.
Chem. Soc. 2004, 126, 438. (b) Lin, W.; Long, L.; Yuan, L.; Cao, Z.;
Chen, B.; Tan, W. Org. Lett. 2008, 10, 5577. (c) Lin, H.; Fan, J.; Wang,
J.; Tian, M.; Du, J.; Sun, S.; Sun, P.; Peng, X. Chem. Commun. 2009,
5904. (d) Lee, J. H.; Lim, C. S.; Tian, Y. S.; Han, J. H.; Cho, B. R. J. Am.
Chem. Soc. 2010, 132, 1216. (e) Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu,
F.; Yang, G. J. Am. Chem. Soc. 2007, 129, 11666. (f) Yao, Z.; Feng, X.;
Li, C.; Shi, G. Chem. Commun. 2009, 5886. (g) Hewage, H. S.; Anslyn,
E. V. J. Am. Chem. Soc. 2009, 131, 13099. (h) Sibrian-Vazquez, M.;
Escobedo, J. O.; Lim, S.; Samoei, G. K.; Strongin, R. M. Proc. Natl.
Acad. Sci. U. S. A. 2010, 107, 551. (i) Zhang, M.; Yu, M.; Li, F.; Zhu, M.;
Li, M.; Gao, Y.; Li, L.; Liu, Z.; Zhang, J.; Yi, T.; Huang, C. J. Am.
Chem. Soc. 2007, 129, 10322. (j) Guo, Z.; Nam, S.; Park, S.; Yoon, J.
Chem. Sci 2012, 3, 2760.
Figure 1. Stacked partial 1H NMR spectra of thiol probe 2 (A)
and conjugate addition product 3 (B) in CDCl3 at 25 °C.
Org. Lett., Vol. 15, No. 1, 2013
217

of BODIPY core is an indication of an increase in the
electron density of the BODIPY core (the signaling unit)
upon conjugate addition.
Once we demonstrated the Michael acceptor reactivity of
the nitroethenyl-BODIPY derivative 2toward the thiol ME,
we have turned our attention to the water-soluble derivative
1. Synthetic route followed is depicted in Scheme 2.
appeared with a maximum at 510 nm. A 15 nm blue shift
with a well-defined isosbestic point at 518 nm is apparent
from the absorption spectrum (Figure 2, left). This
Cys-induced hypsochromic shift reveals the suppression
of ICT process that occurs between donor BODIPY core
and acceptor nitroalkene unit, as predicted. Probe 1 is
essentially nonfluorescent (Φf = 0.056) as evidenced by
emission spectrum (Figure 2, right). Fluorescence intensity
increases up to 20-fold (Figure 2) upon increasing Cys
concentrations (0À400 equiv) with an emission band cen-
tered at 515 nm (λex 500 nm). In agreement with our design
expectations, this fluorescence enhancement is at least
partly due to a reduction in the efficiency of the PeT process
upon the attack of the Cys thiol on probe 1 resulting an
increase in the energy level of the BODIPY centered
LUMO. Under identical conditions, other biological thiols,
such as Homocysteine (Hcy) and Glutathione (GSH),
afforded similar turn on fluorescence responses as well
(Figures S1 and S2, Supporting Information). However,
the response to Cys is much faster than that to Hcy and
GSH. Thus, by controlling the reaction time, exquisite
selectivity for Cys can be obtained (Figure S9, Supporting
Information, and inset in Figure 3).
Scheme 2. Synthesis of the Water-Soluble Thiol Probe 1
Sensing experiments were also carried out in HEPES:
CH3CN (60:40, v/v) and 100% HEPES buffer solutions
(pH 7.2 in both). While 40% acetonitrile solution produces
essentially the same responses as that of 20% acetonitrile
solution, fluorescence response of 1 toward biological
thiols was halved in 100% HEPES solution (Figures S3
and S4, Supporting Information), but the signal is still
strong enough for sensitive and selective detection of thiols
(vide infra).
Gallic acid derived aldehyde 4 was prepared according
to our previous report11 and reacted with 2,4-dimethyl-
pyrrole to furnish 5. Fluorescent dye 5 was formylated via
VilsmeierÀHaack reaction in good yield, and subsequent
condensation in nitromethane in the presence of catalytic
amount of NH4OAc (20 mol %) provided the thiol probe 1
in reasonable overall yields (14%).
Figure2showstheelectronicabsorption and fluorescence
emission response of the thiol probe 1 (2.4 μM) in 50 mM
HEPES:CH3CN (80:20, v/v, pH = 7.20) to increasing
Cysteine (Cys) concentrations (0À400 equiv).
Asa natural extension of thisstudy, wehave investigated
the selectivity of probe 1 toward Cys, Hys and GSH over
potentially competing biologically relevant natural amino
Figure 2. UVÀvis absorption spectra (left) and fluorescence
spectra (right) of the thiol probe 1 (2.4 μM) upon increased
Cys concentrations (0À400 equiv) in 50 mM HEPES:CH3CN
(80:20, v/v, pH = 7.20, λex 500 nm at 25 °C).
Upon addition of Cys, absorption band of free dye 1
centered at 525 nm gradually decreased, and a new band
Figure 3. Fluorescence response of the thiol probe 1 (2.4 μM)
toward biothiols (Cys, Hcy and GSH; 200 equiv each) and other
natural amino acids (400 equiv) in 50 mM HEPES:CH3CN
(80:20, v/v, pH = 7.20, λex 500 nm at 25 °C).
(11) Atilgan, S.; Ekmekci, Z.; Dogan, A. L.; Guc, D.; Akkaya, E. U.
Chem. Commun. 2006, 4398.
218
Org. Lett., Vol. 15, No. 1, 2013

Scheme 3. Red-Emitting Nitroolefin-BODIPY Conjugate Thiol
Probe 7
Table 1. Optical Properties of the Probes 1 and 7
compounda
λabs (nm)
λ
em (nm)
Φfb
εmax
c
Figure 4. Fluorescence spectra of the red-emitting thiol probe 7
(2.4 μM) upon increased Cys concentrations (0À1000 equiv)
in 50 mM HEPES:CH3CN (80:20, v/v, pH = 7.20, λex 600 nm
at 25 °C).
1
7
525
623
540
655
0.056d
0.016e
58 500
51 800
a Data acquired in 50 mM HEPES:CH3CN (80:20, v/v, pH = 7.20),
in dilute solutions. b Relative quantum yields. c Unit: cmÀ1 MÀ1 d Re-
.
ference dye: Rhodamine 6G in water (Φf = 0.95). e Reference dye: Cresyl
violet in methanol (Φf = 0.90).
and meso substituents. Absorption (Figure S7, Supporting
Information) and fluorescence response (Figure 4) of this
red-emitting chromophore 7 were also examined. An equal
magnitude of enhancement response was observed with
the probe 7 and 1 upon incremental Cys additions (0À400
equiv).
In conclusion, two nitroethenyl-BODIPY derivatives (1
and 7) operating at different wavelengths were designed,
synthesized and evaluated for biological thiol (Cys, Hcy
and GSH) sensing in aqueous solutions. Both probes
showed very fast and sensitive responses with color
changes and “turn-on” fluorescence. We are confident that
an understanding of BODIPY photophysics in relation to
PeT efficiency and internal charge transfer would produce
newer and more capable sensors for analytes of interest.
Compounds 1 and 7 are two examples along that promis-
ing road.
acids in HEPES:CH3CN (80:20, v/v) at physiological pH
(7.20). No significant change was observed in both absorp-
tion (Supporting Information) and fluorescence emission
(Figure 3) spectra when natural amino acids other than
cysteine were treated with the probe 1.
Extendingthe sensingrange of theprobe intothered and
near IR would be highly advantageous. To that end, we
made use of now established protocols for Knoevenagel
condensation of the methyl substituted BODIPYs and
aldehydes. As probe 1 was derivatized by reacting with a
trialkoxybenzaldehyde, the absorbance and fluorescence
maxima moved into far red (λabs 623 nm and λem 650 nm)
with enhanced water solubility as a bonus (Scheme 3 and
Table 1).
Synthesis of distyryl-BODIPY 7 was achieved in 3 steps
starting from compound 5 (Supporting Information).
Optical properties of 1 and 7 are given below in Table 1.
Compared to 1, dye 7 has an extended π-conjugation that
causes significant bathochromic shift (≈100 nm in absorp-
tion and ≈125 nm in emission peaks). However, this
extension of conjugation diminished the fluorescence
quantum yield of 7 to approximately one-third of probe 1.
Sensor 7 displayed good water solubility due to the
presence of nine triethyleneglycolic arms on the styryl
Acknowledgment. This research was supported by TU-
BITAK (112T480). We thank Ms. Asli Celebioglu for the
digital photographs.
Supporting Information Available. Methods, experi-
mental procedures, additional spectral data. This materi-
al is available free of charge via the Internet at http://
pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 1, 2013
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