Fluorescence turn-on probe for biothiols: Intramolecular hydrogen bonding effect on the Michael reaction

Article abstract of DOI:10.1016/j.tet.2011.08.002

Weakly fluorescent coumarinyl enones are rapidly transformed into strongly fluorescent molecules through the Michael addition reaction of a thiol group, where an intramolecular hydrogen bond plays a critical role in the reaction rate. The molecular probe

Full text of DOI:10.1016/j.tet.2011.08.002

Tetrahedron 67 (2011) 7759e7762
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Fluorescence turn-on probe for biothiols: intramolecular hydrogen bonding effect
on the Michael reaction
,
Hyun-Joon Ha a, Doo-Ha Yoon b, Seokan Park b, Hae-Jo Kim b
*
a Department of Chemistry and Protein Research Centre for Bio-Industry, Hankuk University of Foreign Studies, Yongin 449-719, Republic of Korea
b Department of Chemistry, Hankuk University of Foreign Studies, Yongin 449-719, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 June 2011
Received in revised form 30 July 2011
Accepted 1 August 2011
Available online 6 August 2011
Weakly fluorescent coumarinyl enones are rapidly transformed into strongly fluorescent molecules
through the Michael addition reaction of a thiol group, where an intramolecular hydrogen bond plays
a critical role in the reaction rate. The molecular probe (3) with an ortho hydroxyl group to a carbonyl
group exhibits a rapid response toward GSH owing to the stabilization of the possible oxyanion in-
termediate by a preferable intramolecular hydrogen bond. Probe 1 with an o-hydroxyl group also showed
a moderately enhanced reaction rate with GSH and soluble in HEPES buffer to exhibit a highly selective
and sensitive fluorescence turn-on response toward biothiols.
Keywords:
Biothiol
Coumarinyl enone
Fluorescence
Hydrogen bond
Michael addition
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Recently we found that the introduction of a preferable hydro-
gen bonding donor unit to the ortho position of a carbonyl group of
A myriad of biological processes are closely related to cellular
thiols, such as cysteine (Cys), homocysteine (Hcy), and -gluta-
a,b-unsaturated enone (3) could accelerate the reaction rate of 3
g
toward 2-mercaptoethanol as fast as several dozen times compared
to 4 without a hydrogen bond.6 This rate acceleration is plausibly
achieved through intramolecular hydrogen bonding to the oxy-
anion of the enolate intermediate. We wonder it is possible to ac-
tivate an enone moiety by employing a hydrogen bonding donor
site to a carbanion of the plausible enolate intermediate in the
Michael reaction. Herein, we report that a Michael reaction is sig-
nificantly affected by an intramolecular hydrogen bonding pattern.
With this aim, a fluorescent probe (1) was prepared and compared
with 3 as a Michael acceptor, where the hydroxyl group of 1 was
introduced to serve as a hydrogen bonding donor to the carbanionic
form of the enolate (Scheme 1). An analogue 2 without a hydroxyl
group was also prepared as a reference molecule.
mylcysteinylglycine (GSH). Cys and Hcy are involved in cellular
growth,1 while GSH in redox homeostasis.2 Alteration in the cel-
lular thiols is also implicated in cancer and AIDS.3 Therefore, se-
lective detection of cellular biothiols is of growing importance.
Many luminescent methods have been extensively pursued due to
their simplicity4 and further reaction-based fluorescent probes
were developed for the selective recognition of the biothiols
through the Michael addition, thiazolidine formation, or disulfide
exchange reaction.5 In aqueous biosphere, however, the reaction is
competitive with the surrounding solvents. The reaction rate is too
dependent on the electronic structure of a probe to quantitatively
rationalize. Hydrogen bond, especially intramolecular hydrogen
bond, is a strong molecular interaction sustainable in an aqueous
environment. Through the systematic investigation of intra-
molecular hydrogen patterns, we can understand the relationship
between the structures of probes and the reaction rates and may
rationally improve the rates. Until now, only a few fluorescent
probes with an intramolecular hydrogen bond have been designed
in viewpoint of the reaction rates in aqueous solvent.
2. Results and discussion
UVevis spectra of probe 1 are dramatically changed when 1 is
treated with GSH in HEPES buffer (Fig. 1). Time-dependent UVevis
spectra of 1 (20 mM) were monitored in the presence of 10 mM GSH.
Upon the addition of GSH,
a maximum peak at 472 nm
(
3
¼1.4ꢀ104 Mꢁ1 cmꢁ1) is hypsochromically shifted to 442 nm with
a pseudo isosbestic point at 470 nm. The formation of 1-GSH is
* Corresponding author. Tel.: þ82 31 330 4703; fax: þ82 31 330 4566; e-mail
address: haejkim@hufs.ac.kr (H.-J. Kim).
apparently complete within 50 min.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.08.002

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H.-J. Ha et al. / Tetrahedron 67 (2011) 7759e7762
Signaling/Reaction/Activation
In order to compare the reaction rates between probes (1e4), we
carried out UVevis kinetics at 25 ꢂC in DMSO/HEPES buffer (Fig. 2),
where 3 and 4 were also soluble. The absorbance changes at
466 nm were monitored upon the addition of GSH (10 mM) to 1 or
Et2N
O
O
O
X
Et2N
O
O
O
X
RSH
R =
2 (10
m
M) to afford the initial rate k (Mꢁ1 sꢁ1) of 8.10ꢀ10ꢁ3 and
1
(X = OH)
(X = H)
SR
2
2.85ꢀ10ꢁ3 for 1 and 2, respectively. Similar rate measurements of 3
and 4 at A485nm have shown the rate constant k to be 2.35ꢀ10ꢁ2 and
9.13ꢀ10ꢁ4 Mꢁ1 sꢁ1 for 3 and 4, respectively (Fig. S4). The kinetic
study has shown that the reaction rate of 1 was enhanced 2.8-fold
compared to 2, while the rate of 3 was enhanced as fast as 26-fold
compared to 4. The rate comparison shows that the oxyanion of 3
can be stabilized by a preferable hydrogen bond and thus the hy-
drogen bond induces significant rate acceleration for 3. However,
the enolate of 1 is structurally difficult to possess such a favorable
hydrogen bond and thus the hydroxyl group induces a little effect
on the reaction rate (Fig. 3).
Et2N
O
O
H3N
CO2
Cys
Cl
NH3
Cysteamine.HCl
3
(X = OH)
(X = H)
O
X
4
Scheme 1. The reaction of conjugated enone probes with a biothiol.
0.6
1
2
3
4
0.4
0.2
0
0
180
360
540
Fig. 1. Time-dependent UVevis spectral changes of 1 (20 mM) with GSH (10 mM) in
HEPES buffer (0.10 M, pH 7.4, 25 ꢂC). Inset: its kinetics.
time/min
Fig. 2. Comparative UVevis kinetics of 1e4 (10
mM) upon the addition of GSH (10 mM)
in DMSO/HEPES buffer (4:1, v/v, 0.10 M, pH 7.4, 25 ꢂC).
Fig. 3. Possible hydrogen bonding patterns of 1 and 3 upon the addition of GSH in HEPES buffer (pH 7.4).

H.-J. Ha et al. / Tetrahedron 67 (2011) 7759e7762
7761
Although probe 1 with an ortho hydroxyl group shows a slight
rate enhancement relative to 2 without a hydrogen bond, it is no-
ticeable that 1 is fairly soluble in HEPES buffer and can be applied
for GSH detection in water. Upon addition of GSH, the fluorescence
intensity of 1 at lem 500 nm (lex 470 nm) is prominently enhanced
and almost saturated around 500 equiv of GSH in HEPES buffer
(0.10 M, pH 7.4). The limit of detection (LOD) of GSH was de-
termined to be 0.14 mM at 3s/m, where s is the standard deviation
of blank measurements and m is the slope obtained from the linear
plot of 1 against GSH (Fig. 4).7 According to the Job’s plot, the
binding stoichiometry between 1 and GSH is observed to be 1:1
(Fig. S5).
Fig. 4. Fluorescence titration curve of 1 (20 mM) at lem/lex 500:470 nm with GSH in
HEPES buffer (pH 7.4). Inset: its linear plot.
Fig. 5. (A) Fluorescence spectra of 1 (20
m
M, lex 470 nm) upon addition of various
amino acids (AA, 500 equiv) in HEPES buffer (0.10 M, pH 7.4) and (B) their competitive
fluorescence intensity changes with GSH (500 equiv) at lem 500 nm.
Encouraged by the UVevis kinetics and the fluorescence ex-
periment, we screened the selectivity of 1 toward the natural amino
acids in HEPES buffer (0.10 M, pH 7.4). Only Cys shows a dramatic
increase (F/Fo 156) in the fluorescence intensity over other amino
acids. Amino acids possessing acidic or basic side chains do not
induce any significant fluorescence changes of 1 (Fig. 5A). GSH,
a main biothiol component in maintaining the cellular redox ho-
meostasis,8 behaves in a similar way as Cys and enhances the
fluorescence intensity of 1 (Fo) (
F
¼0.0003) as much as F/Fo¼168
(F
¼0.012 for 1-GSH).9 Competitive experiments show the consis-
tent selectivity of 1 for GSH. The fluorescence intensity of 1 is al-
most restored to be as big as that of 1-GSH upon the addition of
GSH to the mixtures of 1 and other amino acid (Fig. 5B).
To investigate the reaction mechanism, 1H NMR spectrum of 1
(20 mM in DMSO-d6) was monitored after adding 1.5 equiv cyste-
amine and compared with 1 itself (Fig. S3). The spectra displayed
a highly upfield shift in the phenolic proton (from 10.2 to 9.6 ppm),
along with the disappearance of vinylic protons upon the addition
of cysteamine, which is indicative of the Michael addition reaction
of cysteamine to afford 1-cysteamine adduct. The mass spectral
analysis showed corroborative evidence for the 1-cysteamine ad-
duct: m/z obsd 441 ([MþH]þ, calcd 441.18 for C24H29N2O4S, Fig. S7).
From the fluorescence, NMR, and mass spectral experiments, it
becomes readily apparent that probe 1 selectively reacts with the
thiol-containing amino acids.
The prominent luminescence changes of 1 are observable by the
naked eye. Upon the addition of GSH, the 1-GSH conjugate exhibits
a color change from light yellow to green and dramatically displays
a strong fluorescence, while other natural amino acids do not elicit
the photophysical changes under a portable UV spectroscope ex-
cept Cys (Fig. 6). An amino acid, such as Ser does not induce any
detectable fluorescence changes although it is similar to Cys in
structure.
Fig. 6. Naked-eye visible and fluorescence (lex 365 nm) images of 1 (20 mM) upon
addition of various amino acids (AA, 500 equiv) in HEPES buffer (0.10 M, pH 7.4).
3. Conclusions
We prepared a series of fluorescent probes and compared their
reaction rates with GSH in viewpoint of their intramolecular hy-
drogen bonding patterns. We found that probe 3 with an ortho
hydroxyl group to a carbonyl group exhibits the rapidest response
toward GSH owing to the stabilization of the possible oxyanion
intermediate by a preferable intramolecular hydrogen bond. Probe
1 with an ortho hydroxyl group to the alkene of an enone, which is
prone to stabilize the carbanion form of the enolate intermediate,
showed a moderately enhanced reaction rate with GSH. The probe
(1) exhibited a highly selective and sensitive fluorescence turn-on
response toward biothiols even in 100% HEPES buffer.

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H.-J. Ha et al. / Tetrahedron 67 (2011) 7759e7762
4. Experimental section
J¼6.8 Hz, eNCH2CH3), 1.15 (t, 6H, J¼6.8 Hz, eNCH2CH3). IR (KBr,
cmꢁ1): 2975 (w),1706 (m),1612 (m),1589 (m),15,006 (m),1444 (m),
1343 (m).
4.1. General
All non-aqueous reactions were run in flame-dried glassware
undera positive pressure of nitrogenwith exclusion of moisture from
reagents and glassware using standard techniques for manipulating
air-sensitive compounds. Anhydrous solvents were obtained using
standard drying techniques. Reactions were monitored by analytical
thin-layer chromatography (TLC) performed on pre-coated, glass-
backed silica gel plates. Visualization of the developed chromato-
gram was performed by UV absorbance, phosphomolybdic acid or
iodine. Flash chromatography was performed on 230e400 mesh
silica gel with the indicated solvent systems. Routine nuclear mag-
netic resonance spectra were recorded either on Varian Gemini 200
(200 MHz) or Varian Gemini 400 (400 MHz) spectrometers. Chem-
ical shifts for 1H NMR spectra are recorded in parts per million from
tetramethylsilane with the solvent resonance as the internal stan-
4.2.3. 2-(3-(7-(Diethylamino)-2-oxo-2H-chromen-3-yl)-1-(2-hydrox-
yphenyl)-3-oxopropylthio)ethanaminium (1-cysteamine). 1H NMR
spectra of 1 (20 mM in DMSO-d6) were monitored upon the addition
of cysteamine hydrochloride (1.5 equiv) and the reaction was almost
complete within 2 days. 1H NMR (400 MHz, DMSO-d6):
d 9.61 (s, 1H,
OHPh), 8.42 (s, 1H), 7.92 (br, 3H, NH3Cl), 7.63 (d, 1H, J¼9.2 Hz,
eC(NEt2)CHCHCe), 7.26 (d, 1H, J¼7.6 Hz), 7.02 (t, 1H J¼7.2 Hz), 7.67
(m, 3H, J¼8 Hz), 6.57 (s, 1H), 4.82 (t, 1H, J¼7.2 Hz, cystamineeCHe),
3.69 (dd, 1H, J¼6.8, 17.2 Hz, cystamineeCHCHaCHbe), 3.55 (dd, 1H,
J¼6.8, 17.2 Hz, cystamineeCHCHaCHbe), 3.50 (q, 4H, J¼7.2 Hz,
eNCH2CH3), 3.10 (t, 2H, eSCH2CH2NH3Cl), 3.91 (t, 2H,
eSCH2CH2NH3Cl), 1.14 (t, 6H, J¼7.2 Hz, eNCH2CH3). LRMS (FABþ, m-
NBA): m/z obsd 441 ([MþH]þ, calcd 441.18 for C24H29N2O4Sþ).
dard (CDCl3,
d
7.27 ppm; DMSO-d6 d 2.50 ppm). Data are reported as
Acknowledgements
follows: chemical shift, multiplicity (s¼singlet, d¼doublet, t¼triplet,
q¼quartet, qn¼quintet, m¼multiplet, and br¼broad), coupling
constant in hertz, and integration. Chemical shifts for 13C NMR
spectra are recorded in parts per million from tetramethylsilane us-
ing the central peak of the solvent resonance as the internal standard
This research was supported by the National Research Founda-
tion funded by the Ministry of Education, Science and Technology of
Korea (NRF 2008-C00206) and the Gyonggi Regional Research Cen-
ter (GRRC) program of Gyonggi province (GRRC-HUFS-2011-B03).
(CDCl3, d77.00 ppm). Allspectrawereobtainedwithcompleteproton
decoupling. All fluorescence and UVevis absorption spectra were
recorded in FP 6500 fluorescence spectrometer and Agilent 8453
absorption spectrometer, respectively. Mass spectra were recorded
on G6401A MS-spectrometer. All experiments were carried out with
commercially available reagents and solvents, and used without
further purification, unless otherwise noted.
Supplementary data
Electronic Supplementary data (ESD) available: NMR and mass
spectral data for compounds. Supplementary data associated with
this article can be found, in the online version, at doi:10.1016/
j.tet.2011.08.002.
4.2. Kinetic analysis
References and notes
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The kinetic study of the probes with GSH was performed at
25 ꢂC in DMSO/HEPES buffer (4:1, v/v, 0.10 M, pH 7.4), where all the
probes are soluble. After GSH (10 mM) was added to each of the
probes (10 mM), the absorbance changes at 466 nm (1, 2) or 486 nm
(3, 4) were monitored. The initial rates were measured within 3 h
by applying a kinetic mode in UVevis spectrometry.
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d6):
d
10.21 (s,1H, OH), 8.56 (s,1H), 7.99 (d,1H, J¼16 Hz, eCOCHCHe),
7.94 (d, 1H, J¼16 Hz, eCOCHCHe), 7.67 (d, 1H, J¼8.8 Hz), 7.61 (d, 1H,
J¼6.8 Hz), 7.25 (dt, 1H J¼1.6, 8.4 Hz), 6.92 (d, 1H, J¼8 Hz), 6.86 (t, 1H,
J¼7.6 Hz), 6.78 (dd, 1H, J¼2.4, 8.8 Hz), 6.58 (d, 1H, J¼2.4 Hz), 3.48 (q,
4H, J¼7.2 Hz, eNCH2CH3), 1.14 (t, 6H, J¼7.2 Hz, eNCH2CH3). 13C NMR
(75 MHz, DMSO-d6):
d 186.2, 160.3, 158.6, 157.6, 153.3, 148.7, 138.0,
132.2, 128.8, 124.6, 122.0, 119.9, 116.6, 116.1, 110.5, 108.3, 96.3, 44.8,
12.8. HRMS (FABþ, m-NBA): m/z obsd 364.1544 ([MþH]þ, calcd
364.1549 for C22H22NO4). IR (KBr, cmꢁ1): 3255 (br), 2977 (w), 1705
(m), 1610 (m), 1580 (m), 1496 (m), 1450 (m), 1344 (m).
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ilar procedure was applied as 1 except that benzaldehyde was used
instead of salicylaldehyde. Column chromatographic purification
with EtOAc/Hex (1:1, Rf¼0.50) in 30% yield. 1H NMR (200 MHz,
DMSO-d6): 8.61 (s, 1H), 7.98 (d, 1H, J¼16 Hz), 7.72 (m, 4H), 7.45
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9. It is remarkable that 2 (
tensity upon the addition of GSH (
fluorescence enhancement was observed to be very low compared to that of 1, F/
Fo<4.3 (Fig. S6).
F
¼0.0055) also showed an enhanced fluorescence in-
F
¼0.0091 for 2-GSH). However, the degree of