Proton-Transfer-Based Azides with Fluorescence Off-On Response for Detection of Hydrogen Sulfide: An Experimental, Theoretical, and Bioimaging Study

Article abstract of DOI:10.1021/acs.joc.8b02489

This work describes the synthesis of photoactive proton transfer compounds based on the benzazolic core containing the azide group. The compounds present absorption in the UV region and fluorescence emission in the visible region of the spectra with large

Full text of DOI:10.1021/acs.joc.8b02489

Subscriber access provided by Kaohsiung Medical University
Article
Proton-transfer based azides with fluorescence off–
on response for detection of hydrogen sulfide. An
experimental, theoretical and bioimaging study
Claudia B da Silva, Eduarda S Gil, Fabiano da Silveira Santos, Ana M Moras, Luiza Steffens,
Paulo F. B. Goncalves, Dinara J Moura, Diogo S. Lüdtke, and Fabiano Severo Rodembusch
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02489 • Publication Date (Web): 26 Nov 2018
Downloaded from http://pubs.acs.org on November 27, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Proton-transfer based azides with fluorescence off–on response for detection of
hydrogen sulfide. An experimental, theoretical and bioimaging study
a
a
a
Cláudia Brito da Silva, Eduarda Sangiogo Gil, Fabiano da Silveira Santos, Ana Moira
b
b
a
b
Morás, Luiza Steffens, Paulo Fernando Bruno Gonçalves, Dinara Jaqueline Moura,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
,a
Diogo Seibert Lüdtke, Fabiano Severo Rodembusch*
aInstituto de Química, Universidade Federal do Rio Grande do Sul, UFRGS. Av. Bento
Gonçalves 9500, 91501-970, Porto Alegre, RS, Brazil. Phone: +55 51 3308 9637.
E-mail: rodembusch@iq.ufrgs.br
bLaboratório de Genética Toxicológica, Universidade Federal de Ciências da Saúde de Porto
Alegre, Rua Sarmento Leite, 245, 90050-170, Porto Alegre, Rio Grande do Sul, Brazil
Graphical Abstract:
1
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract. This work describes the synthesis of photoactive proton transfer
compounds based on the benzazolic core containing the azide group. The compounds
present absorption in the UV region and fluorescence emission in the visible region of
the spectra with large Stokes shift due to a phototautomerism in the excited state
(ESIPT). The azide location on the benzazolic structure showed to present a noteworthy
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
role on their photophysics leading to fluorescence quenching. A photophysical study was
performed in the presence of NaHS to evaluate their application as H S sensor. The
2
methodology employed was the reduction of azides to amines using NaHS to mimic
H S, resulting in an off–on response fluorescence mechanism. The observed
2
photophysical features were successfully used to explore the azides as fluorescent
probes in biological media. In addition, DFT and TD-DFT calculations with CAM-
B3LYP/cc-pVDZ and CAM-B3LYP/jun-cc-pVTZ level respectively were performed in
order to understand the photophysics features of azide derivatives, where the main
interest was investigate fluorescence quenching experimentally observed in the azide
derivatives.
Keywords: ESIPT, optical sensor, azides, TD-DFT, fluorescence
Introduction
Hydrogen sulfide (H S) is a small and colorless gas molecule that has a
2
characteristic odor, which may cause irritation to eyes and respiratory system under
prolonged exposure.1,2 H S is produced primarily through the decomposition of organic
2
compounds or as by-products of industries such as oil refining, agriculture, mining,
manufacturing of organophosphorus pesticides, etc.3,4 Although hydrogen sulfide
exhibits chronic and acute toxicity in high concentrations, causing a variety of disease
phenotypes such as Alzheimer's disease, hypertension, liver cirrhosis, Down syndrome
and diabetes,5-8 in low concentrations H S can positively influence physiological effects,
2
including regulation of blood pressure, oxygen sensor, neuromodulation, mitochondrial
energy production and anti-inflammatory action.9-18
19
Several methods of hydrogen sulfide evaluation have been developed, including
electrochemical analysis,20,21 gas chromatography,21,22 and colorimetric and or
fluorometric methods.23-26 Among these methods the emission of fluorescence is
considered one of the most efficient methods due to sensitivity and simplicity and
biological compatibility.27,28 In recent years fluorescent and colorimetric probes have
2
ACS Paragon Plus Environment

Page 3 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
received considerable attention because of their unique advantages such as low cost,
high throughput and naked eye detection.29-31 However, the H S probes still have single
2
detection window and short excitation wavelengths.32 Thus, it is required to design
probes for detection of H S with double detection window with emission in a broad
2
region of the electromagnetic spectrum, as observed for detection of sulfur based
analytes, which present emission at the NIR region.33-36 In this way, a wide variety of
fluorescent probes for this specific detection have been proposed in the literature (Figure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
).37-40 These probes follow different mechanisms, such as azide to amine or nitro to
amine reductions,41-43 R-O cleavage,44,45 S-O cleavage46,47 or double bond addition
reaction.48,49 In this context, in this work is presented the synthesis, photophysical
characterization and application of azide-containing dyes, based on the benzazole core,
for the detection of H S in solution, using NaHS as mimic compound (Figure 1).
2
Figure 1. Examples of azide derivatives described in the literature for H S sensing and
2
azide-containing dyes used in the current work.
The mechanism employed for the H S detection was based on the off-on
2
fluorescence related to the reduction of azides to amines, leading to an increase in
fluorescence intensity. These particular compounds present fluorescence emission since
they are reactive to proton transfer in the excited state (ESIPT) (Scheme 1). In this
process, after radiation absorption, a locally excited enol species (E*) that presents a
proton donor, usually related to an amino or hydroxyl group, is more acidic than in the
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ground state. At the same time, the acceptor group, usually imino/azonitrogen- or
carbonyl-oxygen-containing ring skeleton is also more basic. These photoacidity/basicity
together with distance and angle requirements, allows a faster proton transfer to
produce a keto phototautomer (K*), which decays emitting fluorescence with large
Stokes shift.50-52 In addition, in association with the observed photophysical properties,
these compounds were also evaluated as fluorescent probes in cellular media.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Model for the ESIPT process for a benzazole dye. The asterisk indicates the
excited-state.
Results and Discussion
Photophysical characterization
The absorption spectra of the azides 6-7 and 11-12 are shown in Figure 2. The
relevant data calculated from UV-Vis absorption spectroscopy for all compounds are
presented in Table 1.
4
ACS Paragon Plus Environment

Page 5 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-6
Figure 2. UV-Vis absorption spectra in solution (10 M) of the azides (a) 6, (b) 7, (c) 11
and (d) 12 in different organic solvents.
It can be observed an absorption maxima located in the UV region between 330-
1
3
58 nm with molar absorptivity coefficient values () related to allowed * electronic
transitions. Despite the absorption in the UV region for all studied samples, dyes 6-7
present in general a more intense absorption band located below 300 nm, ascribed to
the benzazole chromophore. This photophysical feature can be related to the difference
of planarity of the dyes,53,54 where the less planar structures 6-7 do not allow a more
effective electronic delocalization among the two π systems (phenolic and benzazolic
rings), which is confirmed taking the molar extinction coefficient values in account. In
addition, it could also be observed that the benzothiazole derivatives 7 and 12 presented
redshifted absorption bands in despite of the benzoxazole ones (6 and 11), related to
the better electron delocalization allowed by the sulfur atom in the benzothiazole core,
as already observed in similar compounds.54
5
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Photophysical data in the ground state of azides 6-7 and 11-12, where abs is
4
-1
-1
the absorption maxima (nm) and ε is the molar absorptivity coefficient (10 M cm ).
Azide
Solvent
abs

Dichloromethane
Ethanol
346
345
345
1.45
0.24
0.45
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Acetonitrile
Dichloromethane
Ethanol
356
358
358
1.23
0.28
0.45
7
Acetonitrile
Dichloromethane
Ethanol
331
330
330
3.13
0.47
2.17
1
1
2
Acetonitrile
Dichloromethane
Ethanol
345
343
343
3.59
0.77
0.95
1
Acetonitrile
The fluorescence emission spectra of the azide derivatives 6-7 and 11-12 in
solution are shown in Figure 3. The emission curves were obtained by exciting the
compounds at the absorption maxima wavelengths ( ). The data from fluorescence
abs
emission spectroscopy are also summarized in Table 2.
It can be observed that the studied azides present a photophysical behavior in the
excited state that is dependent on the azide group position. The compounds 6-7 with the
azide group in the 5’ position are almost non fluorescent in all studied solvents. Despite
the weak fluorescence intensity, a dual fluorescence emission can be observed in
ethanol and dichloromethane, which confirms the conformational equilibrium in solution
in the ground state.50,54 In these compounds, it can be observed an emission located
higher than 500 nm, ascribed to the ESIPT process (Keto* emission). Additionally, a
blue shifted emission, located around 400 nm was observed and related to the normal
emission (Enol* emission). In acetonitrile, both derivatives show only a very weak ESIPT
emission. On the other hand, derivatives presenting the azide group in the 4’ position
(11-12) present a higher fluorescence intensity with both enol (370 nm and 390 nm for
X=O and S, respectively) and keto (470 nm and 500 nm for X=O and S, respectively)
emission at similar location than observed to the analogues 6-7 (Figure 3).
6
ACS Paragon Plus Environment

Page 7 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Table 2. Photophysical data in the excited state of azides 6-7 and 11-12, where em is
-1
-3
the emission maxima (nm), ST is the Stokes shift (nm/cm ) and  (x10 ) is the
FL
fluorescence quantum yield.
em
ST
Azide
6
Solvent
FL
N*
K*
N*
K*
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Dichloromethane
Ethanol
382
-
-
522
503
512
36/2724
176/9745
158/9105
167/9454
1.1
4.7
1.5
-
-
Acetonitrile
Dichloromethane
Ethanol
420
438
420
537
532
551
64/4280
80/5102
62/4123
181/9468
174/9136
193/9784
11.6
3.5
2.0
7
Acetonitrile
Dichloromethane
Ethanol
376
369
376
473
473
474
45/3616
39/3203
46/3707
142/9070
143/9161
144/9206
58.5
24.4
11.5
1
1
2
Acetonitrile
Dichloromethane
Ethanol
399
390
392
500
495
504
54/3923
47/3513
49/3644
155/8986
152/8952
161/9313
7.2
12.1
10.6
1
Acetonitrile
Figure 3. Steady-state fluorescence emission spectra of the azides 6-7 and 11-12 in
-6
solution (10 M) of (a) ethanol, (b) dichloromethane and (c) acetonitrile (Exc./Em. slits
.0 nm/3.0 nm).
5
7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Once again, the benzothiazole derivatives presented redshifted bands in despite
of the benzoxazole analogues, related to the better electron delocalization allowed by
the sulfur atom. It is worth mentioning that it can be observed from the literature that
usually azide compounds present an almost absent or very weak fluorescence emission,
which can be related to a photoinduced electron transfer (PET) mechanism afforded by
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
,
,24,55-57
the –N group.2 3
However, based on the fluorescence results presented in this
3
work, the presence of the –N group seems not to be enough to the expected
3
fluorescence quenching in such derivatives. Moreover, the fluorescence quenching
seems to be significantly favored when the azide group is located in the para position
relative to the hydroxyl group, as shown in compounds 6-7. In this sense, the
experimental results indicate that an additional or even a different mechanism than PET
is tailoring the fluorescence emission in these compounds. Additional photophysical
experiments were also performed at extreme pH for comparison with the data at pH 7.4
(data not shown, see Figures S33-36).
Photophysical response of azides to NaHS
The UV-Vis absorption of the azides 6-7 and 11-12 was investigated upon
-3
addition of NaHS (1.0x10 M) in ethanol/PBS buffer solution (50/50, v/v, pH=7.4) at
room temperature (Figure 4). It can be observed that significant changes took place in
their absorption spectra in presence of NaHS. These compounds showed absorption
peaks around 350 nm. After addition of 10 L NaHS, the absorption intensity of the
azide increases and shifts to longer wavelengths. The observed shift on the absorption
maxima of dyes 6 (~16 nm), 7 (~ 42 nm), 11 (~ 12 nm) and 12 (~ 28 nm) indicates that
there was a change in the chemical structure as the result of the reduction of the azide
54
functional group to an amino group. Although the UV-Vis spectra seems to not present
any clear tendency, since it changes the maxima location, as well as its intensity, it could
-6
-5
be possible to obtain a linear correlation in the range (9.09x10 - 3.03x10 M) between
the absorption with the added amount of NaHS taking the absorption maxima of the
reduced compound into account (insets of Figure 4). This preliminary result using NaHS
already indicates that these azides can be used as probe for the quantitative detection of
H S in solution.
2
8
ACS Paragon Plus Environment

Page 9 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-5
-5
Figure 4. UV-Vis spectra of compounds (a) 6 (2.88x10 M) and (b) 7 (2.98x10 M) and
-5
-5
(
c) 11 (1.98x10 M) and (d) 12 (1.86x10 M) upon addition of different amounts (10-100
-3

L) of NaHS (1.0x10 M) in ethanol/PBS buffer solution (50/50 v/v, pH=7.4). The dash
line is the respective azide in absence of NaHS. The inset presents the respective
correlation between the absorption intensity and NaHS concentration at the absorption
maxima of the reduced compound.
In order to evaluate the azides 6-7 and 11-12 for H S detection by the well-known
2
off-on response presented by azide compounds, fluorescence titrations were also
performed (Figure 5). The azides present low fluorescence intensity prior to the addition
of NaHS. After the addition of different amounts of NaHS a gradual increase in the
fluorescence intensity of the azides was observed, characterizing the off-on response
mechanism. Azides 6-7 and 12 presented an linear relationship between fluorescence
intensity and NaHS concentration,15,57 where azide 6 presented the broader linear range
(10-100 M) if compared to the azides 7 and 12 (20-100 M). This result indicates that
9
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the fluorescence response of these compounds for NaHS can also be used for the
quantitative detection of H S. It is worth mentioning that although azide 11 presented an
2
increase on the fluorescence intensity upon addition of NaHS, a linear response was not
observed, probably due to the dual fluorescence emission that remains present after
addition of 30 L of NaHS.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Fluorescence emission spectra of compounds (a) 6 ( =364 nm, [2.88x10-5
exc
-5
-5
M]) and (b) 7 ( =399 nm, [2.98x10 M]) and (c) 11 ( =334 nm, [1.98x10 M]) and
exc
exc
-5
(
d) 12 ( =371 nm, [1.86x10 M]) upon addition of different amounts (10-100 L) of
exc
-3
NaHS (1.0x10 M) in ethanol/PBS buffer solution (50/50 v/v, pH=7.4) and Exc/Em slits
3.0 nm/3.0 nm. The dash line is the respective azide in absence of NaHS. The inset
presents the respective correlation between the fluorescence intensity and NaHS
concentration at the emission maxima.
Figure 6 presents the azides 6-7 and 12 in ethanol/PBS buffer solution (50/50,
v/v, pH=7.4) under normal light (left) and UV radiation (right). As already observed in
10
ACS Paragon Plus Environment

Page 11 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
their fluorescence emission profile, in absence of NaHS the solutions of 6-7 are non-
fluorescent and 11-12 presents a very weak fluorescence emission, as expected. After
addition of NaHS, the azide compounds are converted to the respective amino
derivatives, which are fluorescent in solution (bottom right). In order to verify the
proposed reaction mechanism, the product of the reaction of compound 7 with NaHS
was analyzed by high resolution electrospray mass spectrometry (ESI-HRMS) and we
have confirmed the structure of compound 5 as the result of the azide-to-amino
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
reduction (M+H m/z 243.0430, see Supporting Information, Figure S41). In addition,
time resolved fluorescence spectroscopy (Figure 7 and Table S3) was also used in order
to investigate the reaction mechanism in these compounds. The full time decay spectra
and the respective residuals are also presented in the ESI (Figures S42-45).
Figure 6. Solutions of azides 6-7 and 11-12 in ethanol/PBS buffer solution (50/50 v/v,
pH=7.4) under normal light (left) and UV radiation (right) in absence (top) and in
presence (bottom) of NaHS.
The fluorescence decay profile of the studied compounds could be fitted by one-
2
exponential decays with good  values. It can be observed that in buffered media the
azide derivative 7 presents in absence of NaHS one single exponential decay with very
short fluorescence lifetime (~0.05 ns) almost overlapping to the instrument response
function (IRF). However, in presence of the NaHS, a commonly employed H S donor,
2
11
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the fluorescence lifetime becomes 3 times longer (~0.15 ns), which can be directly
related to the fluorescence lifetime of the reduced compound 5 at the same buffered
solution (~0.18 ns). A similar value was observed for compound 5 in presence of NaHS
(~0.20 ns), where any chemical reaction were expected. It is believed that the observed
differences to the fluorescence lifetime values can probably be related to the sensibility
of the technique in PBS media.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Fluorescence decays of azide 7 and respective amino derivative 5. [ca. 10-5
M]. IRF=instrument response factor.
Based on the previous photophysical results, the reaction kinetics of the
synthesized azides upon addition of NaHS were also investigated by analyzing the
changes on the UV-Vis (Figure 8) and fluorescence emission (Figure 9) spectra as a
function of reaction time. In Figure 8 is presented the evolution of the absorption maxima
of the azides in time, where it can be observed that each compound behaves differently.
It can be observed that the absorption intensity around 400 nm of azides 6 and 12
decreases after NaHS addition and a new absorption band arises and increases its
intensity at 350 nm. For compounds 7 and 11 the absorption intensity increases and
shifts upon addition of NaHS. In this process, compounds 11 and 12 presents one
isosbestic point located at 363 nm and 377 nm, respectively, suggesting a clean
chemical reduction from azide to amino, as already observed in the literature.15,57
However, compound 6 presents two isosbestic points (370 nm and 416 nm) in despite of
12
ACS Paragon Plus Environment

Page 13 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
its absence for compound 7 indicating that for these derivatives an intricate chemical
transformation is taking place.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Time dependent UV-Vis absorption spectra of the synthesized azides (a) 6
-5
-5
-5
-5
([2.88x10 M]) and (b) 7 ([2.98x10 M]) and (c) 11 [1.98x10 M]) and (d) 12 ([1.86x10
-3
M]) upon addition of different amounts (10-100 L) of NaHS (1.0x10 M) in ethanol/PBS
buffer solution (50/50 v/v, pH=7.4). The black line is the respective azide in absence of
NaHS. The inset presents the respective correlation between the absorption intensity
and NaHS concentration.
Figure 9 presents the evolution of the emission maxima of the azides in time.
Once again, although these compounds presented significant signal changes in
presence of NaHS, it can be observed that each compound shows a particular emission
profile. It can be observed that azides 6 and 7 present very low fluorescence intensity,
which increases significantly after NaHS addition with large Stokes shift. This last
feature is quite interesting for fluorescence imaging applications.58 For compounds 11
and 12 a dual fluorescence emission was already present, which increases its intensity
13
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and blue shift the emission maxima at 375 and 435 nm, respectively. However, in
general the reaction rate almost has no change with the variation of NaHS concentration
after 10 minutes. Compound 6 showed to need longer reaction time to reach the spectra
saturation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 9. Time dependent fluorescence emission spectra of the synthesized azides (a)
-5
-5
6 ( = 356 nm, [2.88x10 M]) and (b) 7 ( = 350 nm, [2.98x10 M]) and (c) 11 ( =
exc
exc
exc
-5
3
34 nm, [1.98x10-5 M]) and (d) 12 ( = 360 nm, [1.86x10 M]) upon addition of
exc
different amounts (10-100 L) of NaHS (1.0x10-3 M) in ethanol/PBS buffer solution
50/50 v/v, pH=7.4). The black line is the respective azide in absence of NaHS. The
(
inset presents the respective e correlation between the fluorescence intensity and NaHS
concentration.
Cell staining
In order to evaluate the efficacy of these probes as H S cell markers, the T98G cells
2
were used, in an experiment model using fixed cells. All four compounds (6-7 and 11-12)
were skilled in cell labeling (Figure 10), demonstrated that the probes are suitable for
14
ACS Paragon Plus Environment

Page 15 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
H S detection, without altering the cells morphology. Considering the 7-AADA nucleus
2
labeling (in blue) and despite that fluorophores containing similar benzazole moieties
stain cell nuclei,59 it seems that the labeling by the different azide probes (in green) is
not specific to any cell structure, resulting in a diffuse cellular marking. It is important to
note, as presented in the literature,32,38,57,60 that cysteine could be regarded as a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
precursor to H S in the cell imaging assays, so in this work the T98G cells were pre-
2
incubated with cysteine and then incubated with synthesized azides. Also, we performed
a control using cells not stained with dyes (7-AAD and azides) and no cells
autofluorescence was observed in time exposure and excitation/emission channels used
(data not shown).
Figure 10. Fluorescence microscopy images of the azides (a) 6, (b) 7, (c) 11 and (d) 12
in T98G fixed cells. The first column shows the fluorescence of cells incubated with the
nuclear dye 7-AAD, central column show the azide compounds and third column the
overlap of the images. Image acquired at 20× as single image field (20× 0.75 NA
objective).
15
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Theoretical calculations
The optimized geometry for the ground and the first excited states of azides 6 and 11 in
dichloromethane are shown in Table 3. The optimized geometries in acetonitrile and
ethanol can be found in Tables S1 and S2 in the SI. It is possible to see, by the
experimental emission spectra values, that there are two emitting species: the enol
tautomer and the keto tautomer. No significant geometry changes were observed for the
enol species when comparing the S0 and S1 states for both azides.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. S and S optimized geometries for azides 6 and 11, computed at the CAM-
0
1
B3LYP/cc-pVDZ level in dichloromethane.
Azide 6
Azide 11
Enol S0
Enol S1
Keto S1
The Figure 11 shows the energies of enol and keto tautomers in ground and first
excited states for azide 11 calculated at the CAM-B3LYP/cc-pVDZ level in acetonitrile.
The enol tautomer of azide 11 is 0.93 eV more stable than the keto tautomer in the S0.
Because of this great energy difference, the energy is absorbed by the enol tautomer, as
experimentally observed. The energy of keto tautomer in the first excited state is only
0.06 eV less stable than enol conformer, and because of this small energy difference,
the enol conformer is easily converted into its tautomer.
16
ACS Paragon Plus Environment

Page 17 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
In Table 4, the NTOs for the first transition of azides 6 and 11 are shown for three
different solvents. We can observe that the NTOs coefficients in the ground and the first
excited states look like π-type orbitals for both azides in all considered solvents. Thus, it
was assumed that π→π* transitions were occurring for both structures. The azide 6
LUMO orbital in dichloromethane is delocalized in the molecule, but in ethanol and
acetonitrile it cannot be observed.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 11. Enol and keto tautomers energies in ground and first excited state for azide
11 computed at the CAM-B3LYP/cc-pVDZ level in acetonitrile.
The calculated photophysical data are shown in Table 5. The CAM-B3LYP/cc-
pVDZ values for absorption and emission wavelength are in good agreement with the
experimental data.
17
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. NTOs orbitals involved in the first transition for azides 6 and 11 computed at
the CAM-B3LYP/jun-cc-pVTZ level in acetonitrile (CH CN), dichloromethane (DCM) and
3
ethanol (EtOH).
Azide
Solvent
S0
S1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CH CN
3
6
DCM
EtOH
CH CN
3
11
DCM
EtOH
18
ACS Paragon Plus Environment

Page 19 of 33
The Journal of Organic Chemistry
1
2
3
4
Table 5. Computed photophysical data at the CAM-B3LYP/jun-cc-pVTZ level for azides
6
an 11 in acetonitrile (CH CN), dichloromethane (DCM) and ethanol (EtOH). Where 
3
abs
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
is the wavelength of absorption, em is the emission wavelength, ƒabs is the absorption
oscillator strength, ƒem is the emission oscillator strength and µ(12) is the transition
dipole moment for the first excitation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Azide
Solvent
abs
6
11
EtOH
DCM
320
EtOH
319
CH CN
DCM
311
CH CN
3
3
319
0.0002
385
310
1.0800
382
309
1.0741
382
ƒabs
0.4082
382
0.0003
385
1.0991
377
em (enol)
ƒem (enol)
em (keto)
ƒem (keto)
µ(12)
0.5862
464
0.6260
469
0.6334
469
1.4464
415
1.1963
417
1.5050
418
0.4649
4.3029
0.4761
0.0026
0.4804
0.0026
0.6854
11.2408
0.7355
11.0075
0.7445
10.9378
However, the main question to be answered in this work, almost absent in the
literature, is "why does azide 11 show more intense fluorescence emission than azide
6?”. In order to answer the question, the transition dipole moment (TDM) values of these
molecules (Table 5) must be taken into account. The TDM for azide 11 is greater than
that of azide 6, therefore, azide 11 has a greater oscillator strength, since the TDM is
proportional to the square of oscillator strength, as shown by the equation (1):
3푒2ℎ
휇2 =
푓

(1)
2
8
휋 푚 
푒
where μ is the dipole moment, e is the electron charge, h is the Planck constant, m is
e
the electron mass,  is the wavenumber and f is the oscillator strength.
We can assume that the azide 11 is able to absorb more energy than azide 6,
thus, a more intense fluorescence emission is observed. This photophysical behavior
can also be related to the TDM value of this molecule in dichloromethane, which is much
higher than in ethanol and acetonitrile.
A quantitative assessment of the local excitation and charge transfer to the
excited states was performed and shows that no charge transfer is present in these
19
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
systems, but only local excitation. The overlap integral between the centroids of charges
(C+ and C-) before and after transition, obtained from the charge transfer diagnostic
index (D ) were computed in acetonitrile and ethanol. The DCT represents the distance
CT
between the centroids of charges obtained before and after the transition. After the
transition, the electronic density tends to be relocated considering the donors and
acceptors groups in the molecule. The results of DCT analysis are shown in Table 6. For
both azides, the centroids of charges before and after transition are localized in the
same region with no displacement between the centroids, excluding the charge transfer
chance for these azides.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. DCT overlap integral between centroids of charges obtained before and after the
transition computed with Multiwfn program for azides 6 and 11 in acetonitrile and
ethanol.
Azide
Solvent
C+
C-
CH CN
3
6
EtOH
CH CN
3
11
EtOH
20
ACS Paragon Plus Environment

Page 21 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Conclusions
In summary, we presented here the synthesis of four novel azide based
benzoxazoles achieved via diazonium salts formation from the respective amino
precursors followed by addition of sodium azide (NaN ) through an unimolecular
3
nucleophilic substitution reaction (S 1). The obtained compounds present absorption
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
maxima located in the UV region (330-360 nm) related to fully spin and symmetry
1
allowed →* electronic transitions. A low intense or almost absent fluorescence
emission bands, tailored by the azide group position were observed in the visible region.
All studied compounds presented significant changes on their photophysics in presence
of NaHS, which allowed apply these compounds as optical sensors for H S in solution.
2
The methodology employed was the reduction of azides to amines using H S, resulting
2
in an off–on response fluorescence mechanism. From the theoretical calculations, it was
observed that the π frontier orbitals are involved in the first transition for azides
1
considered, indicating →* transition is taking place. Furthermore, these excitations
are localized, indication that charge-transfer are not occurring, which has proved by the
DCT descriptor. The fluorescence quenching observed in the azide 6, differently than the
photoinduced electron transfer process (PET), discussed in the literature, and was
explained by the small transition dipole moment for the first transition. Finally, the
fluorescence imaging experiments in T98G cells indicate its potential to probe H S in
2
fixed cells. Since these molecules were efficient for the labeling of biological systems,
further experiments using living cells should be conducted in a next step of this study
and this results will be reported in due course.
Experimental Section
General Information. All reagents were used as received. The solvents were
purchased from commercial sources and purified according to procedures in the
literature.61 The reactions were monitored by Thin-Layer Chromatography (TLC) using
-1
Silica Gel 60 F254. Fourier transform infrared (FTIR) spectra (600-4000 cm ) were
-
1
recorded on a Varian model 640-IR, with a resolution of 4 cm using KBr pellets. Proton
1
13
1
and Carbon nuclear magnetic resonance spectra ( H and C{ H} NMR) were recorded
in CDCl at 300 and 75.5 MHz, respectively. Proton nuclear magnetic resonance spectra
3
(1
carbon nuclear magnetic resonance spectra ( C{ H} NMR) was recorded in CDCl or
H NMR) was recorded in CDCl or DMSO-d at 300 or 400 MHz. Proton-decoupled
3 6
13
1
3
DMSO-d at 75.5 or 101 MHz. The chemical shifts () are reported in parts per million
6
21
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
ppm) relative to TMS (0.00 ppm), and the coupling constants J are reported in hertz
Hz). Melting points were obtained on a QUIMIS model Q340S and are uncorrected. In
(
the photophysical studies, spectroscopic-grade solvents were used. The UV-Vis spectra
were recorded on a Shimadzu UV-2450 spectrophotometer, and the steady-state
fluorescence spectra were measured on a Shimadzu spectrofluorometer model RF-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
301PC. The quantum yield of the fluorescence signals ( ) was obtained with the
FL
dilute optical method using spectroscopic-grade solvents. Quinine sulfate in H2SO4 (0.5
-1
62
mol L ) ( =0.55) was used as the quantum yield standard. Time resolved
FL
fluorescence curves were performed in PBS buffer using an EasyLife V
spectrophotometer (Optical Building Blocks). All measurements were performed at room
temperature. The decay curves were analysed using EasyLife V software (OBB). A
nonlinear least square method was employed for the fit of the decay to a sum of
2
exponentials. The value of  , respective residuals and the autocorrelation function were
used to determine the quality of the proposed fit.
Synthesis. The benzazole precursors 4-5 and 9-10 were obtained by a
condensation reaction between the ortho-substituted anilines 1-2 and the 5- or 4-
o
aminosalicylic acids (3 and 8), respectively in polyphosphoric acid (PPA) at 170 C for 5h
(Scheme S1).63,64 The progress of the chemical reaction was monitored by TLC. The
solution was neutralized with NaHCO (10%), the precipitate was filtered and dried at
3
6
0oC. The purification was performed by column chromatography using dichloromethane
as eluent. The azides 6-7 and 11-12 were synthesized using the reaction of formation of
diazonium salts from the respective aromatic amines 4-5 or 9-10. The first step is the
amino group diazotization by reaction with sodium nitrite (NaNO ) in aqueous HCl.
2
o
During this step, the mixture temperature was maintained at 0 C in order to stabilize the
resulting diazonium salt, which could improve reaction yields. After the formation of the
diazonium salt, sodium azide (NaN ) was added and the reaction to obtain the
3
respective azides takes place through an unimolecular nucleophilic substitution reaction
(S 1). After 2h under stirring, the final solution was neutralized with NH OH, extracted
N
4
with ethyl acetate and concentrated under reduced pressure to yield the desirable
azides in satisfactory yields, which were used without further purification.
General procedure for the synthesis of precursors 4-5 and 9-10. An
equimolar mixture of o-aminophenol (1) (2.8 g, 26.12 mmol) or o-aminothiophenol (2)
22
ACS Paragon Plus Environment

Page 23 of 33
The Journal of Organic Chemistry
1
2
(2.76 mL, 26.12 mmol) and 5-aminosaliylic acid (3) or 4-aminosaliylic acid (8) (4.0 g,
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
6,12 mmol) in polyphosphoric acid (60 mL) was stirred at 170°C for 5 h. After cooling,
the mixture was poured onto ice under vigorous stirring. The solution was neutralized
o
with NaHCO . The precipitate was filtered, washed with water and dried at 60 C. The
3
solid was purified by column chromatography using dichloromethane as eluent.54
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2-(5’-Amino-2’-hydroxyphenyl)benzoxazole (4). Yield: 71% (0.294 g). Melting
-1
point: 173-175ºC: FTIR (KBr, cm ): 3405 (as NH ), 3250 (s NH ), 1630 and 1548 (
2
2
1
C=C). H NMR (300 MHz, DMSO-d )  (ppm): 10.42 (s, 1H, OH), 7.81 (m, 2H), 7.44 (m,
6
13
1
2H), 7.26 (d, 1H, J=2.3 Hz), 6.84 (m, 2H), 4.96 (s, 2H). C{ H} NMR (75.5 MHz, DMSO-
d6)  (ppm): 162.8, 149.3, 148.6, 141.7, 139.6, 125.6, 125.2, 121.3, 119.0, 117.6, 110.9,
110.4, 109.6.
2-(5’-Amino-2’-hydroxyphenyl)benzothiazole (5). Yield: 53% (0.271 g). Melting
-1
point: 196-198ºC. FTIR (KBr, cm ): 3497 (as N-H), 3325 (s N-H), 3000 (
C-H),
arom
1
1620 and 1470 ( C=Carom). H NMR (300 MHz, DMSO-d ) ( ppm): 10.72 (s, 1H, OH),
6
8.12-7.40 (m, 4H, H1, H2, H3, H4), 7.37-7.36 (d, 1H, J=2.7 Hz), 6.85-6.83 (d, 1H, J=8.7
13
1
Hz), 6.77-6.76 and 6.74-6.73 (2d, 1H, J=2.7 Hz and J=8.8 Hz), 4.8 (s, 2H). C{ H} NMR
75.5 MHz, DMSO-d )  ppm): 166.3, 151.6, 147.8, 141.5, 134.0, 126.4, 125.0, 121.9,
(
6
121.8, 119.9, 117.8, 117.6, 112.0.
2-(4’-Amino-2’-hydroxyphenyl)benzoxazole (9). Yield: 44% (0.182 g). Melting
-1
point: 226-228ºC. FTIR (KBr, cm ): 3493 (as N-H), 3386 (s N-H), 3000 (arom C-H),
1
1620 and 1470 ( C=Carom). H NMR (400 MHz, CDCl )  (ppm): 10.95 (s, 1H), 7.76-7.71
3
(m, 1H), 7.63-7.58 (m, 1H), 7.42-7.36 (m, 3H), 6.99 (d, 1H, J=4.1 Hz), 6.87 (dd, 1H,
13
1
J=8.7Hz and J=2.8 Hz), 3.56 (s, 2H). C{ H} NMR (101 MHz, CDCl )  (ppm): 163.6,
3
160.6, 151.7, 148.9, 140.4, 128.6, 124.6, 124.3, 118.5, 110.2, 107.3 ,101.5, 101.5.
2-(4’-Amino-2’-hydroxyphenyl)benzothiazole (10). Yield: 33% (0.169 g).
-1
Melting point: 211-213ºC. FTIR (KBr, cm ): 3473 (as N-H), 3376 (s N-H), 3000 (
arom
1
C-H), 1620 and 1470 ( C=Carom). H NMR (400 MHz, CDCl )  (ppm): 12.66 (s, 1H),
3
7.90 (d, 1H, J=8.1 Hz), 7,86 (d, 1H, J=7.9 Hz), 7.49-7.43 (m, 2H), 7.38-7.33 (m, 1H),
6.34 (d, 1H, J=2.2 Hz), 6.28 (dd, 1H, J=8.4 Hz and J=2.3 Hz), 4.05 (s, 2H). 13C{ H}
1
23
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NMR (101 MHz, CDCl )  (ppm): 169.6, 160.1, 152.2, 150.9, 132.3, 130.0, 126.4, 124.6,
3
121.4, 121.3, 108.5, 107.2, 101.7.
General procedure for the synthesis of azides 6-7 and 11-1. In a 50 mL round
bottom flask under an ice bath containing 2.0 mmol of the benzazole precursors 4-5
452 and 485 mg, respectively) or 9-10 (452 and 485 mg, respectively) and a HCl:H O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2
(3:1) solution (15 mL) it was added dropwise a NaNO (0.166 g, 2.4 mmol) solution
2
(
1mL). The reaction mixture was stirred under ice bath for 30 min. After this time, NaN3
was added (0.260 g, 4.0 mmol). The reaction mixture was stirred at room temperature
for 2h. After this time, the solution was neutralized with NH OH, extracted with ethyl
4
acetate (3x20 mL) and concentrated under reduced pressure to yield the respective
product.
2-(5’-Azido-2’-hydroxyphenyl)benzoxazole (6). Yield: 79% (0.464 g). Melting
o
-1
point: 143-145 C. FTIR (KBr, cm ): 3056 (
C-H), 2117 ( -N=N=N), 1553 and 1471
arom
1
(
 C=Carom). H NMR (400 MHz, CDCl )  (ppm): 11.37 (s, 1H), 7.77-7.72 (m, 1H), 7.67-
3
13
1
7
.61 (m, 2H), 7.45-7.39 (m, 2H), 7.16-7.07 (m, 2H). C{ H} NMR (101 MHz, CDCl ) 
3
(ppm): 161.9, 156.0, 149.1, 139.8, 131.5, 125.8, 125.2, 124.4, 119.4, 118.9, 116.6,
+
111.2, 110.8. HRMS (ESI-TOF) m/z: [M+H] Calcd for C H N O 253.0726; Found
13
9
4
2
253.0723.
2-(5’-Azido-2’-hydroxyphenyl)benzothiazole (7). Yield: 70% (0.456 g). Melting
o
-1
point: 147-149 C. FTIR (ATR, cm ): 3086 ( O-H), 2098 ( -N=N=N), 1613 and 1513 (
1
C=Carom). H NMR (400 MHz, CDCl )  (ppm): 12.43 (s, 1H), 7.99 (d, 1H, J=8.1 Hz), 7.91
3
(d, 1H, J=7.9 Hz), 7.56-7.49 (m, 1H), 7.46-7.40 (m, 1H), 7.26 (d, 1H, J=2.5 Hz), 7.11-
1
7
.05 (m, 2H). 13C{ H} NMR (101 MHz, CDCl )  (ppm): 161.9, 156.0, 149.1, 139.8,
3
131.5, 125.8, 125.2, 124.4, 119.4, 118.9, 116.6, 111.2, 110.8. HRMS (ESI-TOF) m/z:
+
[M+H] Calcd for C H N OS 269.0497; Found 269.0477.
13
9
4
2-(4’-Azido-2’-hydroxyphenyl)benzoxazole (11). Yield: 90% (0.464 g). Melting
-1
point: 111-112ºC. FTIR (ATR, cm ): 3056 ( O-H), 2118 ( -N=N=N), 1644 and 1573 (
1
C=Carom). H NMR (300 MHz, CDCl )  (ppm): 11.60 (s, 1H), 7.98 (d, 1H, J=7.7 Hz), 7.67
3
13
1
(
d, 2H, J=7.9 Hz), 7.40 (s, 2H), 6.79 (s, 1H), 6.68 (d, 1H, J=7.2 Hz). C{ H} NMR (101
MHz, CDCl )  (ppm): 162.3, 159.9, 149.0, 139.9, 128.5, 125.4, 125.1, 119.1, 110.9,
3
24
ACS Paragon Plus Environment

Page 25 of 33
The Journal of Organic Chemistry
1
2
+
1
10.6, 107.6, 107.4. HRMS (ESI-TOF) m/z: [M+H] Calcd for C H N O 253.0726;
13
9
4
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Found 253.0726.
2-(4’-Azido-2’-hydroxyphenyl)benzothiazole (12). Yield: 89% (0.456 g). Melting
-1
point: 132-134ºC. FTIR (ATR, cm ): 3077 ( O-H), 2117 ( -N=N=N), 1653 and 1573 (
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
C=Carom). H NMR (400 MHz, CDCl )  (ppm): 12.79 (s, 1H), 7.96 (d, 1H, J=7.7 Hz), 7.88
3
(d, 1H, J=7.5 Hz), 7.61 (d, 1H, J=8.4 Hz), 7.54-7.49 (m, 1H), 7.45-7.38 (m, 1H), 6.74 (d,
1H, J=2.2 Hz), 6.59 (dd, 1H, J=8.4 Hz and 2.2 Hz). 13C{ H} NMR (75 MHz, CDCl ) 
1
3
(ppm): 162.3, 159.9, 149.0, 145.2, 139.9, 128.5, 125.4, 125.1, 119.1, 110.9, 110.6,
+
107.5, 107.3. HRMS (ESI-TOF) m/z: [M+H] Calcd for C H N OS 269.0497; Found
13
9
4
269.0493.
H S detection. The study of detection of H S with the azides 6-7 and 11-12 was
2
2
-5
performed using a solution of ethanol with concentration in the range of 10 M. To these
solutions were added different amounts (0-100 M) of NaHS (1.0x10-3 M) in
ethanol/PBS buffer solution (50/50, v/v, pH = 7.4) at room temperature.
Theoretical calculations. The geometry optimizations and vibrational
frequencies were evaluated using DFT and the vertical transitions were computed using
TD-DFT (Time-Dependent Density Functional Theory). The CAM-B3LYP65 functional
was chosen to perform all the calculations. This functional was chosen because it shows
good results for a series of applications, especially in TD-DFT calculations, as reported
in the literature.66-68 All the geometry optimizations were carried out using the Dunning
69
cc-pVDZ basis set, and a jun-cc-pVTZ basis set was used in TD-DFT calculations. The
use of jun- basis functions was because it is necessary diffuse functions for a good
description of excited states. These basis function were chosen since they have a lower
computational cost than aug- type base functions, since jun- basis functions are
70
constructed by reducing diffuse functions of aug- basis functions. Since the sulphurous
analogues 7 and 12 presented similar photophysical features than the oxygenated ones,
the calculations were performed only for the azides 6 and 11. The solvent effect was
considered in all calculation using PCM (Polarizable Continuum Model), and the
solvents considered were acetonitrile (CH CN), dichloromethane (DCM) and ethanol
3
(
EtOH).71 The NTOs (Natural Transition Orbital) were computed. NTOs drastically
simplify the orbital analysis by providing a compact representation of the transition
25
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
density matrix.72 All the calculations described above were computed using the
73
Gaussian 16 package. In order to verify if the molecules undergo charge transfer, the
analysis with DCT descriptor74 has been carried out using Multiwfn program. All images
75
were rendered with Chemcraft software.76
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cell Culture. The glioblastoma multiform T98G cells were acquired by American

Type Culture Collection (ATCC). The cells were grown in Dulbecco’s modified Eagle’s
medium (Gibco™ DMEM, Life Technology, NY, USA) supplemented with 10% heat-
inactivated fetal bovine serum (Gibco™ FBS, Life Technology, NY, USA), 100 units ml-1
-1
penicillin and 100 g ml streptomycin at 37°C in a humidified atmosphere with 5% CO .
2
Fluorescence Response in Biological System. In order to investigate the
4
azides as optical sensors, cells (1.0 x 10 cells) grown on 96-well plates were fixed in
paraformaldehyde (3.7%), washed with PBS, permeabilized in Triton X-100 (0.01%) and
incubated with cysteine (100 M, 30 min, 37°C). Then, the cells were washed with PBS
and incubated with the azides 6-7 or 11-12 (10 mM) for 30 min at 37°C. The 7-AAD was
used to counteract the nucleus, according to manufacturer's recommendations (BD
Biosciences, San Diego, CA). After, the cells were imaged in InCell analyzer 2200 (GE
Healthcare Life Sciences, Piscataway, NJ, USA) at 390/18 nm excitation and 432.5/48
emission to azides and 597/45 excitation and 542/27 nm emission to 7-AAD.
Supporting Information:
The Supporting Information is available free of charge on the ACS Publications
website. Copies of NMR spectra for all compounds, additional photophysical and
theoretical data.
Acknowledgements
The authors would like to acknowledge FAPERGS (17/2551-0000968-1), CNPq
and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) -
Finance Code 001 for the financial support. Special thanks for M.Sc. Luana Silva for
HRMS data.
26
ACS Paragon Plus Environment

Page 27 of 33
The Journal of Organic Chemistry
1
2
3
4
5
References
1. Evans, C. L. The Toxicity of Hydrogen Sulphide and other Sulphides. Quart. J. Exp.
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Physiol. 1967, 52, 231-248.
2. Zhi, G.; Chen, G.; Zeng, G.; Li, Z.; Chen, A.; Wang, J.; Jiang, L. Fluorescence
Chemosensors for Hydrogen Sulfide Detection in Biological Systems. Analyst 2015, 140,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1772-1786.
3. Li, J.; Yin, C.; Huo, F. Chromogenic and Fluorogenic Chemosensors for Hydrogen
Sulfide: Review of Detection Mechanisms Since the Year 2009. RSC Adv. 2015, 5,
2191-2206.
4. Pandey, S. K.; Kim, K. H.; Tang, K. T. A Review of Sensor-Based Methods for
Monitoring Hydrogen Sulfide. Trends Anal. Chem. 2012, 32, 87-99.
. Lippert, A. R. Designing Reaction-Based Fluorescent Probes for Selective Hydrogen
Sulfide Detection. J. Inorg. Biochem. 2014, 133, 136-142.
. Guidotti, T. L. Hydrogen Sulfide: Advances in Understanding Human Toxicity. Int. J.
Toxicol. 2010, 29, 569-581.
. Abe, K.; Kimura, H. The Possible Role of Hydrogen Sulfide as an Endogenous
5
6
7
Neuromodulator. J. Neurosci. 1996, 16, 1066-1071.
8. Mustafa, A. K.; Sikka, G.; Gazi, S. K.; Steppan, J.; Jung, S. M.; Bhunia, A. K.;
Barodka, V. M.; Gazi, F. K.; Barrow, R. K.; Wang, R.; Amzel, L. M.; Berkowitz, D. E.;
Snyder, S. H. Hydrogen Sulfide as Endothelium-Derived Hyperpolarizing Factor
Sulfhydrates Potassium Channels. Circ. Res. 2011, 109, 1259-1268.
9. Papapetropoulos, A.; Pyriochou, A.; Altaany, Z.; Yang, G.; Marazioti, A.; Zhou, Z.;
Jeschke, M. G.; Branski, L. K.; Herndon, D. N.; Wang, R.; Szabó, C. Hydrogen Sulfide is
an Endogenous Stimulator of Angiogenesis. Proc. Natl. Acad. Sci. USA 2009, 106,
21972-21977.
1
0. Szabó, C.; Papapetropoulos, A. Hydrogen Sulphide and Angiogenesis: Mechanisms
and Applications. Br. J. Pharmacol. 2011, 164, 853-865.
1. Olson, K. R.; Dombkowski, R. A.; Russel, M. J.; Doellman, M. M.; Head, S. K.;
1
Whitfield, N. L.; Madden, J. A. Hydrogen Sulfide as an Oxygen Sensor/Transducer in
Vertebrate Hypoxic Vasoconstriction and Hypoxic Vasodilation. J. Exp. Biol. 2006, 209,
4011-4023.
12. Peng, Y. J.; Nanduri, J.; Raghuraman, G.; Souvannakitti, D.; Gadalla, M. M.; Kumar,
G. K.; Snyder, S. H.; Prabhakar, N. R. H S Mediates O Sensing in the Carotid Body.
2
2
Proc. Natl. Acad. Sci. USA 2010, 107, 10719-10724.
27
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 33
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3. Lee, M.; Schwab, C.; Yu, S.; McGeer, E.; McGeer, P. L. Astrocytes Produce the
Antiinflammatory and Neuroprotective Agent Hydrogen Sulfide. Neurobiol. Aging 2009,
0, 1523-1534.
3
14. Fu, M.; Zhang, W.; Wu, L.; Yang, G.; Li, H.; Wang, R. Hydrogen Sulfide (H S)
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Metabolism in Mitochondria and Its Regulatory Role in Energy Production. Proc. Natl.
Acad. Sci. USA 2012, 109, 2943-2948.
15. Xiang, K.; Liu, Y.; Li, C.; Tian, B.; Tong, T.; Zhang, J. A. A Colorimetric and
Ratiometric Fluorescent Probe With a Large Stokes Shift for Detection of Hydrogen
Sulfide. Dyes Pigm. 2015, 123, 78-84.
16. Eto, K.; Asada, T.; Arima, K.; Makifuchi, T.; Kimura, H. Brain Hydrogen Sulfide is
Severely Decreased in Alzheimer's Disease. Biochem. Biophys. Res. Commun. 2002,
293, 1485-1488.
17. Kamoun, P.; Belardinelli, M. C.; Chabli, A.; Lallouchi, K.; Chadefaux-Vekemans, B.
Endogenous Hydrogen Sulfide Overproduction in Down Syndrome. Am. J. Med. Genet.
A 2003, 116, 310-311.
18. Fiorucci, S.; Antonelli, E.; Mencarelli, A.; Orlandi, S.; Renga, B.; Rizzo, G.; Distrutti,
E.; Shah, V.; Morelli, A. The Third Gas: H S Regulates Perfusion Pressure in Both the
2
Isolated and Perfused Normal Rat Liver and in Cirrhosis. Hepatology 2005, 42, 539-548.
1
9. Hartle, M. D.; Pluth, M. D. A Practical Guide to Working with H2S at the Interface of
Chemistry and Biology. Chem. Soc. Rev., 2016, 45, 6108-6117.
0. Lawrence, N. S.; Davis, J.; Jiang, L.; Jones, T. G. J.; Davies, S. N.; Compton, R. G.
2
The Electrochemical Analog of the Methylene Blue Reaction: A Novel Amperometric
Approach to the Detection of Hydrogen Sulfide. Electroanalysis 2000, 12, 1453-1460.
21. Spilker, B.; Randhahn, J.; Grabow, H.; Beikirch, H.; Jeroschewski, P. New
Electrochemical Sensor for the Detection of Hydrogen Sulfide and Other Redox Active
Species. J. Electroanal. Chem. 2008, 612, 121-130.
22. Bérubé, P. R.; Parkinson, P. D.; Hall, E. R. Measurement of Reduced Sulphur
Compounds Contained in Aqueous Matrices by Direct Injection Into a Gas
Chromatograph with a Flame Photometric Detector. J. Chromatogr. A 1999, 830, 485-
489.
23. Kato Jr., E. T.; Yoshida, C. M. P.; Reis, A. B.; Melo, I. S.; Franco, T. T. Fast
Detection of Hydrogen Sulfide using a Biodegradable Colorimetric Indicator System.
Polym. Int. 2011, 60, 951-956.
28
ACS Paragon Plus Environment

Page 29 of 33
The Journal of Organic Chemistry
1
2
3
24. Yu, F.; Han, X.; Chen, L. Fluorescent Probes for Hydrogen Sulfide Detection and
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Bioimaging. Chem. Commun., 2014, 50, 12234-12249.
25. Ren, T. B.; Xu, W.; Zhang, Q. L.; Zhang, X. X.; Wen, S. Y.; Yi, H. B.; Yuan, L.;
Zhang, X. B. Enhancing the Anti-Solvatochromic Two-Photon Fluorescence for Cirrhosis
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Imaging by Forming a Hydrogen-Bond Network. Angew. Chem. Int. Ed. 2018, 57, 7473-
7477.
26. Chen, W.; Pacheco, A.; Takano, Y.; Day, J. J.; Hanaoka, K.; Xian, M. A Single
Fluorescent Probe to Visualize Hydrogen Sulfide and Hydrogen Polysulfides with
Different Fluorescence Signals. Angew. Chem. Int. Ed. 2016, 55, 9993-9996.
27. Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J. H.; Yoon, J. Cyanine-
Based Fluorescent Probe for Highly Selective Detection of Glutathione in Cell Cultures
and Live Mouse Tissues. J. Am. Chem. Soc. 2014, 136, 5351-5358.
28. Gao, M.; Wang, R.; Yu, F.; You, J.; Chen, L. A Near-Infrared Fluorescent Probe for
the Detection of Hydrogen Polysulfides Biosynthetic Pathways in Living Cells and in
Vivo. Analyst 2015, 140, 3766-3772.
29. Sun, S. G.; Qiao, B.; Jiang, N.; Wang, J.; Zhang, S.; Peng, X. Naphthylamine-
Rhodamine-Based Ratiometric Fluorescent Probe for the Determination of Pd2+ Ions.
Org. Lett. 2014, 16, 1132-1135.
30. Wang, X.; Guo, Z.; Zhu, S.; Tian, H.; Zhu, W. A Naked-Eye and Ratiometric Near-
Infrared Probe for Palladium via Modulation of a π-Conjugated System of Cyanines.
Chem. Commun. 2014, 50, 13525-13528.
31. Pu, S. Z.; Ma, L. L.; Liu, G.; Ding, H. C.; Chen, B. A Multiple Switching Diarylethene
2
+
with a Phenyl-Linked Rhodamine B Unit and Its Application as Chemosensor for Cu .
Dyes Pigm. 2015, 113, 70-77.
32. Bae, S. K.; Heo, C. H.; Choi, D. J.; Sen, D.; Joe, E.; Cho, B. R.; Kim, H. M. A
Ratiometric Two-Photon Fluorescent Probe Reveals Reduction in Mitochondrial H S
2
Production in Parkinson’s Disease Gene Knockout Astrocytes. J. Am. Chem. Soc. 2013,
135, 9915-9923.
33. Gao, M.; Wang, R.; Yu, F.; You, J.; Chen, L. Imaging and Evaluation of Sulfane
Sulfur in Acute Brain Ischemia using a Mitochondria-Targeted Near-Infrared Fluorescent
Probe. J. Mater. Chem. B 2018, 6, 2608-2619.
34. Gao, M.; Wang, R.; Yu, F.; You, J.; Chen, L. Evaluation of Sulfane Sulfur Bioeffects
via a Mitochondria-Targeting Selenium-Containing Near-Infrared Fluorescent Probe.
Biomaterials 2018, 160, 1-14.
29
ACS Paragon Plus Environment