A turn on ESIPT probe for rapid and ratiometric fluorogenic detection of homocysteine and cysteine in water with live cell-imaging

Article abstract of DOI:10.1016/j.tetlet.2013.11.055

5(Benzothiazol-2-yl)-4-hydroxyisophthalaldehyde (BHI), an intense ESIPT containing molecule in mixed media loses its properties due to resonance-assisted H-bond (RAHB) in absolute water. Due to resonance-assisted H-bond the o-aldehyde is more reactive than the other one. With addition of cysteine/homocysteine into this solution the o-aldehyde group gets transformed into thiazolidine/thiazine ring, respectively, and the phenolic proton becomes free enough for transfer to nitrogen of the benzothiazole ring in excited state, that is, the ESIPT of BHI is turned on. Thus we can detect cysteine/ homocysteine in water as well as in live cells.

Full text of DOI:10.1016/j.tetlet.2013.11.055

Tetrahedron Letters 55 (2014) 490–494
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A turn on ESIPT probe for rapid and ratiometric fluorogenic detection
of homocysteine and cysteine in water with live cell-imaging
a
a
a
a
Shyamaprosad Goswami a, , Abhishek Manna , Sima Paul , Avijit K. Das , Prasanta K. Nandi ,
⇑
Anup Kumar Maity b, Partha Saha b
a Bengal Engineering and Science University, Shibpur, Howrah 711103, India
b Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata 700064, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history:
5(Benzothiazol-2-yl)-4-hydroxyisophthalaldehyde (BHI), an intense ESIPT containing molecule in mixed
media loses its properties due to resonance-assisted H-bond (RAHB) in absolute water. Due to resonance-
assisted H-bond the o-aldehyde is more reactive than the other one. With addition of cysteine/homocys-
teine into this solution the o-aldehyde group gets transformed into thiazolidine/thiazine ring, respec-
tively, and the phenolic proton becomes free enough for transfer to nitrogen of the benzothiazole ring
in excited state, that is, the ESIPT of BHI is turned on. Thus we can detect cysteine/homocysteine in water
as well as in live cells.
Received 15 August 2013
Revised 13 November 2013
Accepted 16 November 2013
Available online 22 November 2013
Keywords:
Ratiometric sensor
Homocysteine and cysteine sensor
ESIPT
Ó 2013 Elsevier Ltd. All rights reserved.
Cell-imaging
Resonance assisted hydrogen bond
Inter- and intramolecular proton transfers are fundamental
reactions in chemistry and biology that have been broadly investi-
gated.1 A broad class of molecules that contain an H-chelated ring
exhibits both in the gas phase and in nonpolar solvents fast ex-
cited-state intramolecular proton transfer (ESIPT).2 ESIPT com-
pounds have also drawn much attention due to their potential
applications in optical devices that may take advantage of the sali-
ent properties such as the ultra-fast reaction rate and extremely
large fluorescence Stokes shift.3 Riedle and co-workers4 have nicely
explored this area by taking 2-(20 hydroxyphenyl)benzothiazole
(HBT) as a standard moiety. Taking this advantage of HBT, many
researchers have designed anion and cation sensors with ESIPT
mechanism.5
We also take HBT as an ESIPT containing fluorophore with the
addition of aldehyde group for taking advantage of RAHB, that is,
resonance-assisted H-bond introduced by Gilli et al.6 in 1989. This
also helps in selective and ratiometric detection of cysteine (Cys)
and homocysteine (Hcy) which are essential biological molecules
relevant to the growth of cells and tissues in living systems.7 Cys
deficiency is involved in many syndromes, for instances, slow
growth in children, liver damage, skin lesions, and weakness.8
Hcy has also been linked to increased risk of Alzheimer’s disease,9
inflammatory bowel disease, and osteoporosis.10
CHO
OH
hexamine
S
S
CHO
AcOH, tol.
reflux
N
OH
N
Scheme 1. Synthetic outlook.
6
6
5
5
1.6x10
1.2x10
8.0x10
4.0x10
BHI in mixed
aqueous media
BHI in water
0.0
400
450
500
550
600
650
Wavelength (nm)
⇑
Corresponding author. Fax: +91 3326682916.
E-mail address: spgoswamical@yahoo.com (S. Goswami).
Figure 1. Solvent dependent fluorescence response of BHI (c = 2.0 ꢀ 10ꢁ5 M).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.11.055

S. Goswami et al. / Tetrahedron Letters 55 (2014) 490–494
491
CHO
Therefore, the detection and discrimination of thiol-containing
molecules in biological samples are of great importance. Due to
simplicity, sensitivity, and efficiency of fluorescence methods11 de-
sign of fluorescent receptors is desirable for potential applications.
In the past few years, various fluorescent chemodosimeter probes
for biothiols based on different mechanisms have been devel-
oped.12 From those, a few are also capable of displaying high selec-
tivity over other amino acids by turning on the fluorescence at
neutral pH in pure water.13 But a ratiometric approach toward
Cys and Hcy at neutral pH in pure water considering both the
strong H-bonding phenomena, that is, RAHB and ESIPT is still a
challenge. It is well known that, ratiometric probes allow measure-
ment at two different wavelengths, which could provide a built-in
correction for environmental effects and improve the detection
sensitivity.14 Thus, a chemodosimeter exhibiting greatly changed
emission intensity with ratiometric manner is ideal for sensing.
Thus considering all the above facts, herein we report a HBT
based probe, 5(benzothiazol-2-yl)-4-hydroxyisophthalaldehyde
(BHI) for highly selective and sensitive detection of Cys/Hcy in pure
water and cells. The probe (BHI) was prepared according to our
previously reported procedure (Scheme 1).15 It has a HBT moiety
as a fluorescence signaling unit and salicyldehyde functionality
as a reaction unit. Both Cys and Hcy react with the carbonyl group
of BHI to afford a five or six-membered ring.
CHO
O
RAHB
> in water
H
S
H
S
O
O
N
O
H
N
H
H
CHO
CHO
ESIPT
= in mixed
media
H
S
S
O
O
N
O
O
N
H
H
Scheme 2. Probable solvent dependent isomers of BHI.
)
(
SH
CHO
HOOC
n
CHO
OH
Hb
N
NH2
Ha
S
S
Cys, n=1
Hcy, n=2
S
)
(
n
OH
N
O
N
H
COO-
In our previous work,15 we see that BHI is a strong ESIPT con-
taining agent in mixed aqueous media (CH3CN/H2O = 1:1). Upon
irradiation (kex = 344 nm), BHI generates the excited-state
Scheme 3. Reaction mode of Cys/Hcy with BHI.
1.4x106
1.2x106
1.0x106
8.0x105
6.0x105
4.0x105
2.0x105
BHI
,,+ 10µl Cys
,,+ 20µl Cys
,,+ 30µl Cys
,,+ 40µl Cys
,,+ 50µl Cys
,,+ 60µl Cys
,,+ 70µl Cys
,,+ 90µl Cys
,,+110µl Cys
,,+1300µl Cys
,,+150µl Cys
,,+175µl Cys
,,+200µl Cys
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Y = 126620X + 0.4164
R2=.997
5.0x10-6 1.0x10-5 1.5x10-5 2.0x10-5 2.5x10
-5
0.0
0.0
[Cys]
400
600
650
450Wav5e0le0ngth 5(5n0m)
Figure 2. Fluorescence spectra of BHI (c = 2.0 ꢀ 10ꢁ5 M) in the presence of 1 equiv Cys (c = 2.0 ꢀ 10ꢁ4 M, total 200
corresponding ratiometric response (I521/I436) of BHI with the addition of Cys.
ll in various amounts) in buffered-H2O (at pH 7.4) and the
BHI
1.4x10 6
,,+ 10µl Hcy
,,+ 20µl Hcy
1.2x10 6
,,+ 30µl Hcy
,,+ 40µl Hcy
,,+ 50µl Hcy
1x106
1x106
1x106
1x106
9x105
8x105
7x105
6x105
5x105
1.0x10 6
8.0x10 5
6.0x10 5
4.0x10 5
,,+ 60µl Hcy
,,+ 70µl Hcy
,,+ 90µl Hcy
,,+110µl Hcy
,,+1300µl Hcy
,,+150µl Hcy
,,+175µl Hcy
,,+200µl Hcy
521nm
R2=0.9969
436nm
R2=.9946
2.0x10 5
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
0.0
[Hcy]/µM
400
450
500
550
600
650
Wavelength (nm)
Figure 3. Fluorescence spectra of BHI (c = 2.0 ꢀ 10ꢁ5 M) in the presence of 1 equiv Hcy (c = 2.0 ꢀ 10ꢁ4 M, total 200
ll in various amounts) in buffered-H2O (at pH 7.4) and the
corresponding intensity plot as a function of [Hcy] at two different wavelengths.

492
S. Goswami et al. / Tetrahedron Letters 55 (2014) 490–494
intramolecular proton transferred (ESIPT) tautomers (the keto
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
forms) which show fluorescence more strongly at a longer wave-
length (at 521 nm) compared to the phenol forms (at 436 nm).
The enol isomer, which is lower in energy than the keto isomer
in the electronic ground state, undergoes the proton transfer reac-
tion upon excitation to the excited state in mixed aqueous media.
But in absolute water the most intense fluorescence peak of BHI at
436 nm confirms the hampered ESIPT of HBT (Fig. 1). Probably in
the presence of polar protic solvent, the proton of hydroxyl group
is strongly bonded with the nearest carbonyl group (RAHB). Thus
the particular proton of BHI whose transfer (ESIPT) is the main
cause of longer wavelength fluorescence is engaged in other pur-
poses (Scheme 2).
Tyr Lys
Ilu
Leu
Cys
Ser
ThrArg
Met
Trp
Phe
Ala ValPro
GSH
Glu
His
Hcy
Gly
BHI
In this circumstance, when we add Hcy/Cys, the hampered
ESIPT of BHI is regained rapidly through the RAHB. Due to strong
H-bonding the carbonyl group becomes very electron deficient
which is readily converted into thiazoline or thiazolidine in the
presence of Hcy or Cys, respectively, (Scheme 3), as it is well
known that the reaction of aldehydes with the N-terminal group
of Cys forms thiazolidines.16 Thus the proton of the phenol moiety
is free enough to move to nitrogen of bezothiazole moiety which is
an essential condition for ESIPT of BHI (Scheme 3).
Figure 4. Ratiometric response of BHI toward 1 equiv of various amino acids with
their ‘naked eye’ response under UV-light.
To measure the fluorescence response of BHI for amino acids,
fluorescence titration was carried out with a series of amino acids
Figure 5. Partial 1H NMR spectrum of BHI and its adduct with Cys in DMSO-d6.
(a)
(c)
(b)
(d)
Figure 6. Energy minimized structure of (a) BHI in enol form, (b) BHI in keto form,
(c) BHI-CN in enol form, and (d) BHI-CN in keto form from DFT calculation.
Enol form
Keto form
CHO
CHO
*
*
1.4x106
1.2x106
1.0x106
8.0x105
6.0x105
4.0x105
Hb
Hb
S
S
ESIPT
S
S
)n
(
)n
(
N
O
N
N
O
N
H
H
H
H
COOH
COOH
521 nm
344 nm
CHO
CHO
Hb
Hb
S
)n
N
S
(
S
)n
N
S
O
H
N
(
0
1
2
3
4
5
6
7
8
9
H
O
H
N
Time (min.)
COOH
H
COOH
Figure 7. Time-dependent change of fluorescence intensity of BHI (20
lM) at
436 nm (in red) and 521 nm (in black) in the presence of Cysteine (1.0 equiv at a
Figure 8. Molecular orbitals and electronic contributions of the relevant excitations
time) at pH 7.4 in H2O.
for BHI and BHI-Hcy in water with the corresponding 4-level diagram.

S. Goswami et al. / Tetrahedron Letters 55 (2014) 490–494
493
Figure 9. Fluorescence images of HeLa cells incubated with 50
l
M of the receptor (BHI) in the absence (a and b) and in the presence (e and f) of 50
l
M of cysteine.
Corresponding differential interference contrast (DIC) images (c and g) and merge images (d and h) of the cells are shown.
for example Cys, Hcy, N-Boc-Cys, Met, Ser, Thr, Lys, Trp, Asp, Pro,
Glu, Leu, Ile, Gly, Ala, His, Phe, Val, Arg, Tyr, and GSH under biolog-
ical pH at room temperature.
are much more stable than the corresponding form of BHI (about
1.79 ꢀ 106 kJ/mol) which also suggests the rapid response of Hcy
to BHI (Figs. 6 and 7).
Only with the addition of Hcy/Cys, the emission at 521 nm is in-
creased with a ‘naked eye’ color change from blue to green (under
UV-light) with a well-defined iso-emissive point at 488 nm in
water (HEPES buffer, pH 7.4) and gives a better ratiometric re-
sponse over others (Fig. 4). Glutathione, a thiol containing amino
acid also reacts with BHI, but the lower response than Cys/Hcy
makes BHI a specific probe for these amino acids. Probably forma-
tion of the more stable product (with cyclic structure) is the main
reason for this type of preferable reaction over others. Fluorescence
In order to determine the membrane permeability of BHI and its
ability to specifically bind to the Cys in living cells, HeLa cells were
first incubated with the cysteine molecules followed by the addi-
tion of the receptor. A control incubation of the cells with BHI only
was also carried out. As shown in Figure 9, the cells showed intense
green fluorescence in FITC channel when they were treated with
Cys molecule followed by BHI. Nuclei of the cells stained in DAPI
showed deep blue color. No fluorescence was observed when the
cells were not treated with the cysteine molecule. The result
clearly established that the receptor (BHI) could permeate the plas-
ma membrane of the cells and give specific fluorescence only in the
presence of Cys molecule.
In conclusion, we have successfully shown an ESIPT containing
receptor (BHI) which lost its ESIPT characteristics (due to RAHB) in
water and is recovered (through RAHB) with the addition of cys-
teine/homocysteine over other amino acids in absolute aqueous
solution with live cell-imaging. BHI exhibits a unique rapid ratio-
metric fluorescence change in the presence of Hcy/Cys demonstrat-
ing its excellent selectivity over other amino acids.
changes were monitored by using a 20 lM solution of BHI in water
sharply, followed by the ratiometric decrease in the emission at
436 nm, which indicated that the chemical reaction between the
following thiol containing amino acids and the aldehyde group
interrupted the RAHB and the original ESIPT of BHI is regenerated
strongly (Figs. 2 and 3).
The linear ratiometric response (I521/I436) of BHI with increasing
concentration of Cys also makes it a unique probe for it (Fig. 2). The
fluorescence intensity of BHI is also changed linearly with in-
creased concentration of Hcy between 0.2
different wavelengths (Fig. 3). From the plot, it is clear that
0.7 M of Hcy is enough for ratiometric response of BHI at physio-
lM and 1.6 lM at two
l
Acknowledgments
logical condition.
To confirm that the thiazolidine ring was formed by the reaction
of Cys with the aldehyde group of BHI, we investigated the 1H NMR
spectrum of BHI upon addition of Cys and compared it with that of
the probe itself. Complete conversion of BHI to its Cys adduct was
accomplished by the addition of Cysto BHI in DMSO-d6 (Fig. 5). As
the aldehyde proton (Ha) at around d 13 ppm disappeared, the thi-
azolidine methine proton (Hb) at around d 5.41 ppm newly ap-
peared and the aromatic protons are up field shifted. Under the
same conditions, Hcy also reacted similarly with BHI and formed
the six-membered ring of thiazinane. Interestingly, the aldehyde
proton para to the hydroxyl group remains almost unchanged in
the NMR spectrum. The mass spectrum (ESI MS) of the Cys-adduct
shows peaks at m/z 387.04 possibly due to BHI+Cys+H+ ions, which
also proves a single mononuclear addition of cysteine to BHI, m/z
284.03 (BHI+H+) (Supplementary data).
We thank D.S.T. and CSIR (Govt. of India) for financial support.
A.M. and A.K.D. acknowledge CSIR and S.P. acknowledges UGC for
providing a fellowship.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.11.
055.
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