Fate of protected HBT based chemodosimeters after undergoing deprotection: Restoration of ESIPT or generation of emissive phenoxide?

Article abstract of DOI:10.1016/j.chemphys.2019.01.008

The -OH group protected 2-(hydroxyphenyl) benzothiazole (HBT) based ratiometric chemodosimeters have previously been reported to undergo regeneration of excited state intramolecular proton transfer (ESIPT) process upon deprotection. In the current work it is demonstrated for the first time that generation of anion of HBT leads to the optical changes in these types of chemodosimeters rather than regeneration of HBT by using a Hydrazine specific chemodosimeter based on substituted HBT.

Full text of DOI:10.1016/j.chemphys.2019.01.008

Accepted Manuscript
Fate of protected HBT based chemodosimeters after undergoing deprotection:
restoration of ESIPT or generation of emissive phenoxide?
Arghyadeep Bhattacharyya, Subhash Chandra Makhal, Nikhil Guchhait
PII:
S0301-0104(18)31201-1
DOI:
https://doi.org/10.1016/j.chemphys.2019.01.008
CHEMPH 10275
Reference:
To appear in:
Chemical Physics
Received Date:
Revised Date:
Accepted Date:
31 October 2018
22 December 2018
6 January 2019
Please cite this article as: A. Bhattacharyya, S.C. Makhal, N. Guchhait, Fate of protected HBT based
chemodosimeters after undergoing deprotection: restoration of ESIPT or generation of emissive phenoxide?,
Chemical Physics (2019), doi: https://doi.org/10.1016/j.chemphys.2019.01.008
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Fate of protected HBT based chemodosimeters after undergoing deprotection: restoration
of ESIPT or generation of emissive phenoxide?
*
Arghyadeep Bhattacharyya, Subhash Chandra Makhal, and Nikhil Guchhait
Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata-700009, India
*
Corresponding author. Tel.: +913323508386; Fax: +913323519755.
Abstract
-OH protected 2-(hydroxyphenyl) benzothiazole (HBT) based ratiometric chemodosimeters
have previously been reported to undergo regeneration of excited state intramolecular proton
transfer (ESIPT) process upon deprotection. In the current work it is demonstrated for the first
time that generation of anion of HBT leads to the optical changes in these types of
chemodosimeters rather than regeneration of HBT by using a Hydrazine specific
chemodosimeter based on substituted HBT.
Keywords
HBT; ESIPT; chemodosimeter; ratiometric; anion

1
. Introduction
Since the epoch making work by Weller, excited state intramolecular proton transfer [1] (ESIPT)
process has gained tremendous attention from chemists due to dual emission and large Stokes’
shift. The difference in the nature of ESIPT in various systems also provide excellent
opportunities for theoretical chemists to propose newer models which aid in uncovering the
mechanism behind ESIPT processes [2-4]. However, due to easy synthetic route, 2-(2’-
hydroxyphenyl) benzothiazole (HBT) backbone can be tailored at will to provide a number of
ESIPT derivatives and hence HBT is widely used by analytical, organic as well as inorganic
chemists to design many beautiful sensors. Dual emission with appreciably distant bands [5-8]
observed in bare HBT has prompted chemists to design brilliant optical chemosensors for
detecting large range of analytes. These are of two types, non-chemodosimetric [9] and
chemodosimetric [10]. While the former type undergoes complexation/acid-base type reaction
with analyte(s); the latter undergoes reaction with analyte(s) to furnish optical signal. Judicious
protection of the phenolic –OH moiety in HBT results in generation of protected form (PF) of
the former. Analyte(s) undergoes chemical reaction with PF to generate the HBT in free form
(FF). Using this approach many receptors have been designed for detecting analytes ranging
from cations, anions as well as neutral analytes [11-23]. Interestingly, almost all reports have
shown that generation of FF is associated with a typical ‘blue-fluorescence’ when observed
under the UV-light and a ratiometric response is also observed during this reaction with the
emission maxima at ~450-470 nm (Table 1). This new peak in emission profile as well as the
colour observed under the UV-light is assigned to be due to the regeneration of ESIPT. In
addition, two perturbing factors are also pertinent: a) no spectral comparison between FF and the
parent HBT molecule from which the PFs were generated. Thus, instead of a ‘point-by-point’

correlation of the peak representing the so-called ‘ESIPT’ form, the entire assignment was done
by assuming that cleavage of PF leads to ESIPT. b) All the studies were carried out in polar
protic/mixture of polar protic and aprotic solvent systems where ESIPT from HBT-based probes
are reported to be disfavoured [6]. At this juncture, an interesting self-contradicting report further
increased our confusion. Goswami et al. reported the ESIPT regaining of a probe in presence of
amino acids homocysteine and cysteine by increment of a band at ~520 nm [24]. The same group
reported the regeneration of ESIPT at an emission maxima of 462nm [17] associated with a blue
2
-
luminescence in presence of H S/S . In both cases, the parent skeleton was HBT. In order to
2
verify the claim of ESIPT restoration, a similar model compound 2-(benzo[d]thiazol-2-yl)-4-
bromophenyl benzoate (PHBT) was hence designed from to detect Hydrazine in aqueous
methanol as there is increasing need for selective detection of hydrazine owing to its many
beneficial and detrimental aspects [25-27]. PHBT shows a ratiometric response in presence of
Hydrazine and can successfully detect Hydrazine in real water samples. The color change
observed under UV-light was blue as previous reports. However, by careful spectroscopic
comparison of PHBT-Hydrazine ensemble with the parent compound 2-(benzo[d]thiazol-2-yl)-
4
-bromophenol (UHBT) revealed that the anionic form of UHBT (AUHBT) was responsible for
the spectral results instead of regeneration of ESIPT as claimed by previous works. To remove
any possible ambiguity due to presence of bromine substitution in PHBT, a thorough literature
survey was done which revealed that for unsubstituted HBT also, anion is generated. Thus, the
present work finds its own merit for establishing the true reason behind the optic response of
protected HBT derivatives upon deprotection.

em
max
Table 1. Reported 
of protected HBT based chemodosimeters
em
Receptor(reference) Analyte detected  max(nm)
BTB(10)
APBT(9)
FBBP(11)
CS1(8)
Hydrazine
458
455
462
460
470
-
BO3
2
-
H S/S
2
2
+
Cu
PHBT(current work) Hydrazine
2
2
5
. Experimental
.1. Reagents
-bromosalicylaldehyde, 2-aminothiophenol, benzoyl chloride, metal perchlorate salts and
Tetrabutylammonium salts of anions were purchased from Merck and used as received. All
solvents were of spectroscopic grade purchased from Spectrochem.
2
.2. Apparatus
Steady state electronic absorption and fluorescence spectra were recorded on a Hitachi UV-Vis
1
(
Model U-3501) spectrophotometer and Perkin Elmer LS55 Fluorimeter respectively. H NMR
spectra were recorded on a Bruker Advance 300 spectrometer, where chemical shifts (δ in ppm)
were determined with respect to Tetramethyl silane (TMS) as the internal standard. Mass
spectrum was recorded on Waters Xevo G2-S Q TOF mass spectrometer.

2
.3. Synthetic Scheme
In short, UHBT was synthesized (Scheme 1) by reacting 5-bromosalicyladehyde (2.0 mmol), 2-
aminothiphenol (2.0 mmol) and molecular iodine (1.0 mmol) in 20 mL methanol and filtering the
greenish white solid obtained after 5 hr stirring at room temperature. The NMR spectra of UHBT
are provided in Fig. S1. In the next step, 1.0 mmol of benzoyl chloride was added to a solution of
UHBT (1.0 mmol) in dry DCM followed by adding equivalent amount of triethylamine. The
entire reaction mixture was stirred overnight and then filtered, followed by acidic work-up of the
filtrate. Subsequent evaporation of DCM layer provided PHBT as a grey solid in 59% yield
(Scheme S1). The NMR and ESI-MS spectra are provided in Fig.S2-S5.
Br
CHO
OH
NH2
SH
MeOH
S
Br
N
I2, stirring
OH
2
-(benzo[d]thiazol-2-yl)-4-bromophenol(UHBT)
Br
S
S
N
Cl
O
Br
DCM
N
Et N, stirring
3
OH
O
O
2
-(benzo[d]thiazol-2-yl)-4-bromophenyl benzoate (PHBT)
Scheme 1. Synthetic outline for PHBT.

3
. Results and discussions
Receptor PHBT was synthesized by a two step reaction starting from 5- bromosalicylaldehyde.
The detailed synthesis and characterization has been provided in the supplementary information
(Fig. S2-S5) and the purity was hence verified.
3
.1. Response of PHBT as a probe for Hydrazine
To a 30 µM solution of PHBT in aqueous methanol (3: 7, v/v), 100 equivalents of various
amines (Fig. 1) were added. Addition of only Hydrazine and 2, 4-DNP resulted in color changes.
However, when observed under UV-light, the vial containing Hydrazine only showed a strong
blue emission. When the UV-spectrum was recorded for all the amines utilized for naked eye
color change along with various metals and anions, no interference was observed, even from 2,
4
-DNP. Only Hydrazine addition resulted in a new profile with bands at 290, 345 and 400 nm
respectively (Fig. 2).Thus, the color change in case of addition of 2, 4-DNP was due to the
yellow color in itself and it could be stated that PHBT is selective towards Hydrazine. When
excited at 300 nm, a 5 µM solution of PHBT itself shows an emission maxima centered at ~380
nm. Upon addition of Hydrazine, the aforesaid profile diminished and a new peak of much
higher intensity appeared at 475 nm. Such a response was not obtained upon addition of other
amines as well as metals or anions (ESI, Fig. S6).

Fig. 1. Color Change of PHBT (far left; 30µM) under ambient light (top) and UV-light (bottom).
Analytes added (from left) hydrazine, phenyl hydrazine, 2, 4-dinitrophenyl hydrazine,
orthophenylene diamine, aniline, ethylene diamine, diethylenetriamine, urea and tris (each 100
equivalent)
Fig. 2. a) UV-Vis response of PHBT (10 µM) towards various amines including Hydrazine,
cations and anions; b) UV-Vis titration of PHBT (10 µM) with Hydrazine (0-20 equivalent).

Fig. 3. Fluorimetric titration of PHBT (5 µM) with Hydrazine (0-10 equivalents).
The above obtained results proved the potential of PHBT to be a selective ratiometric probe for
Hydrazine. To find out the mechanism of Hydrazine detection, ESI-MS experiments were
conducted. A suspension of PHBT in methanol was treated with hydrazine and stirred overnight.
The mixture thus obtained was subjected to ESI-MS. The prominent signal appeared for UHBT
+
(
[M+H ] calc. 305.9588 and 307.9588; obtained 305.9484 and 307.9482) (Fig. 4). In another
experiment, Hydrazine was added instantly to a solution of PHBT and was subjected to ESI-MS.
Two peaks were found corresponding to masses of PHBT as well as UHBT which clearly prove
that UHBT is formed as a result of reaction of PHBT with Hydrazine. A very small signal was
obtained having a mass corresponding to that of PHBT+N H . The small intensity proves that
2
4
this is the intermediate of the reaction of PHBT with Hydrazine (Fig. 5).

Fig. 4. ESI-MS of PHBT-Hydrazine adduct.

Fig. 5. ESI-MS of an instantly prepared mixture of PHBT and Hydrazine.
Furthermore, NMR spectra were recorded for PHBT-N H ensemble (ESI, Fig. S7, S8). Hence it
2
4
could be concluded that parent compound 2-(benzo[d]thiazol-2-yl)-4-bromophenol (UHBT) was
generated from PHBT upon addition of Hydrazine and the latter is thus a chemodosimeter for
Hydrazine.

3
.2. Practical application of PHBT
At this stage, we sought out to find a practical application of PHBT for Hydrazine detection. We
prepared a drinking water sample spiked with Hydrazine (20 µM) and added the same to an
aqueous methanolic solution of PHBT. Although no naked eye color change was observed, a
blue luminescence was observed under UV-light; ensuring the practical utility of PHBT (Fig. 6).
Fig. 6. Detection of Hydrazine in drinking water by PHBT observed under UV-light (right).
3
.3. Comparison of PHBT-N H ensemble with UHBT
2 4
Our observations were consistent with other existing reports up to this point. Instead of
concluding that ESIPT is restored upon deprotection of PHBT, we conducted a comparative
spectral study of PHBT-Hydrazine ensemble with the parent compound UHBT. A thorough
spectral investigation of the parent compound UHBT was first conducted (Fig.7, 8) for that
purpose. The UV-Vis profile of UHBT furnished two bands at 290 nm and 340 nm; the latter
corresponded to the coupling between benzothiazole and the hydroxyphenyl ring [28]. In DMSO
an additional band appeared at 415 nm (Fig. 7). To ascertain the nature of this band, base was

added to a solution of UHBT in acetonitrile, as in acetonitrile no major contribution is present
apart from the two strong absorbing bands. Upon addition of base, a band was generated at 403
nm, indicating anion formation upon base addition. Hence, the third band in DMSO was
attributed to anion generation of UHBT. The red shift in the anionic band in DMSO compared to
acetonitrile could be attributed to its high stabilisation in DMSO (dielectric constant value of
~47). The basic nature of DMSO also contributes to the easy formation of anion in the said
solvent. UHBT showed single emission band only in non-polar solvents (only ESIPT operative;
em

max~520 nm) whereas dual emission was observed for polar protic as well as aprotic solvents
em
(
( max=390 and 470 (DMSO)/515(ACN) nm). For methanol a triple emission was observed
with an additional band at ~470 nm. The peak at ~520 nm was hence due to ESIPT and local
emission of UHBT was responsible for the band at 390 nm by comparing similar bands reported
in literature for HBT [5]. The band at ~470 nm was also observed upon addition of base to
UHBT in acetonitrile (Fig. 9). Hence, the band at ~470 nm was attributed to the anionic form of
UHBT [6]. In polar protic solvent like methanol, the phenolic proton undergoes efficient
intermolecular hydrogen bonding with methanol. Due to the aforementioned intermolecular
hydrogen bond formation, the intramolecular hydrogen bond between the phenolic –OH proton
and the N atom of benzothiazole moiety is ruptured. Consequently, the proton in UHBT
becomes labile and thus the emission spectrum contains three bands. The hindrance in
occurrence of ESIPT in polar solvents due to intermolecular hydrogen bonding has also been
reported previously by theoretical chemists also by the aid of meticulous computational methods
[
2-5]. Had ESIPT been restored after adding Hydrazine to PHBT, a band at ~520 nm would
naturally be expected to appear. But from Fig. 3 it was obvious that no peak or even a shoulder
was generated at 520 nm. The peak at 470 nm was assigned to be the anionic form of UHBT

(AUHBT). A comparison of the UV-Vis and Fluorescence profiles in Fig. 9 gives a clear idea of
the generation of AUHBT from PHBT after addition of Hydrazine. A less rigorous experiment
provided an easy to apprehend yet solid reasoning behind formation of AUHBT. The colours of
UHBT solutions in various solvents were observed under UV-light (Fig. 10). The colour
appeared to be yellowish green in almost all solvents except DMSO where it changes to blue due
to anion generation. A similar blue colour was observed when base was added to UHBT (Fig.3).
Recalling that addition of Hydrazine to PHBT furnishes a similar blue colour (Fig. 1), it could be
stated that this colour was typical of the anion of the HBT concerned. Thus, the deprotection of
PHBT affords AUHBT than UHBT itself. Emission maxima for ESIPT and anionic emission of
a series of HBT based derivatives have been tabulated in Table 2. A perusal of the data given in
Table 2 indicates that substitution effect has little influence on the positions of bands
corresponding to anionic and proton transferred form of HBT derivatives. At this juncture, the
report by Potter et al. proved to crucial [8] where it was clearly stated that the 460 nm band
observed in unsubstituted HBT is due to formation of anion in polar solvents. In short, in HBT
derivatives the ESIPT emission corresponds to a wavelength greater than 500 nm and the
anionic form has emission maxima between ~450-480 nm. The bromine substitution in our
synthesized model PHBT hence does not affect the generality of our conclusions. Thus, we
confirmed that the claim of ESIPT restoration from cleavage of PF were consequences of
correct observation but improper explanation and it could be stated in general that deprotection
of protected HBT based compounds affords the anion of the HBT concerned.

Table 2. Tabulated representation of max for ESIPT and anionic form in various HBT
derivatives.
em
em
Structure of ESIPT

of

of
max
max
active compound(reference number)
ESIPT form
anionic form
~500 nm
~ 530 nm
~509 nm
~460 nm
S
N
OH
(5)
~465 nm
S
N
OH
(4)
Not
Provided
(4)
~
515 nm
540 nm
~470 nm
~480 nm
(3)
~
(
4)

Fig. 7. a) UV-Vis spectra of UHBT in various solvents. b) Base effect observed in acetonitrile.

Fig. 8. a) Fluorescence Spectrum of UHBT in various solvents ( =340 nm); b) Solid state
ex
Fluorescence spectrum of UHBT ( =340 nm); c) UHBT+1 drop acetonitrile in acetonitrile.
ex

Fig. 9. Comparative a) UV-Vis and b) Fluorescence profiles of UHBT, UHBT+base, PHBT and
PHBT+hydrazine ensembles.
Fig. 10. Base effect of UHBT under UV-light for a) methanol; b) acetonitrile and c) colour of
UHBT observed under UV light in solvents of varying polarity.

4
. Conclusions
In conclusion, the present study negates a long existing misleading explanation behind the
ratiometric response of protected HBT based chemodosimeters upon deprotection. The observed
blue emission observed during the ratiometric response is not due to ESIPT generation but due
to anion formation in polar solvent(s). We supported our claim by synthesizing a novel protected
bromo substituted HBT derivative (PHBT) and conducted thorough spectroscopic comparison
with its unprotected analogue (UHBT). In addition, PHBT can be utilised for detecting
Hydrazine in drinking water.
Acknowledgement
AB and SM would like to thank CSIR and UGC for granting fellowships. AB would like to give a
heartfelt thanks to Mr. Sudipto Debnath, University of Calcutta for valuable suggestions regarding the
syntheses. NG thanks DST, India (Project No. EMR/2016/004788) for financial support.
Appendix A. Supplementary Information.
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Supplementary Information
Fate of protected HBT based chemodosimeters after undergoing deprotection: restoration
of ESIPT or generation of emissive phenoxide?
*
Arghyadeep Bhattacharyya, Subhash Chandra Makhal and Nikhil Guchhait
Table of Contents:
1
2
3
4
5
6
. PMR and CMR spectra of UHBT in CDCl3.
. PMR spectrum of PHBT in CDCl3.
. CMR spectrum of PHBT in CDCl3.
. FTIR spectrum of PHBT.
. ESIMS of PHBT
. Fluorimetric response of PHBT (10 µM) in presence of various amines, hydrazine,
cations and anions
7
8
. PMR spectrum of PHBT-N H adduct in DMSO-d .
2 4 6
. CMR spectrum of PHBT-N H adduct in DMSO-d .
2
4
6

1
. PMR and CMR spectra of UHBT in CDCl3.
Fig.S1 a) PMR and b) CMR spectra of UHBT in CDCl3.

2
. PMR spectrum of PHBT in CDCl3.
Fig.S2 PMR spectrum of PHBT in CDCl3.

3
. CMR spectrum of PHBT in CDCl3.
Fig. S3 CMR spectrum of PHBT in CDCl3.

4
. FTIR spectrum of PHBT.
Fig. S4 FTIR spectrum of PHBT.
. ESIMS of PHBT
5
+
Fig. S5 ESIMS of PHBT; calculated for [M+Na ] =431.9560 and 433.9549.

6
. Fluorimetric response of PHBT (10 µM) in presence of various amines, hydrazine,
cations and anions
Fig. S6 Fluorimetric response of PHBT (10 µM) in presence of various amines, hydrazine,
cations and anions.

7
. PMR spectrum of PHBT-N H adduct
2 4
Fig. S7 PMR spectrum of PHBT-N H adduct in DMSO-d .
2
4
6
8
. CMR spectrum of PHBT-N H adduct
2 4
Fig.S8 CMR spectrum of PHBT-N H adduct in d -DMSO.
2
4
6

Graphical abstract
Fate of protected HBT based chemodosimeters after undergoing deprotection: restoration
of ESIPT or generation of emissive phenoxide?
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Arghyadeep Bhattacharyya, Subhash Chandra Makhal, and Nikhil Guchhait
Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata-700009, India