Nitric Oxide Sensing through 1,2,3,4-Oxatriazole Formation from Acylhydrazide: A Kinetic Study

Article abstract of DOI:10.1021/acs.joc.8b02110

A simple molecular probe displays highly selective turn-on response toward NO by the unprecedented NO-induced formation of a 1,2,3,4-oxatriazole ring exhibiting no interference from various endogenous biomolecules including DHA, AA, etc. Kinetics of the reactions between NO and the probe provide a mechanistic insight into the formation of 1,2,3,4-oxatriazole which showed that, though initially 1,2,3,4-oxatriazole is formed and extractable in solid form, it exists in equilibrium with the ring opened azide form which ultimately hydrolyzed and converted to carboxylic acid and nitrate. The reaction displays second-order dependence on [NO] and first-order on [Probe]. The probe is water-soluble, cell permeable, and noncytotoxic and appropriates for live cell imaging. This constitutes the first report where there is a direct evidence of NO-induced ring closing reaction of an acyl hydrazide moiety leading to the formation of 1,2,3,4-oxatriazole.

Full text of DOI:10.1021/acs.joc.8b02110

Subscriber access provided by University of Sunderland
Article
Nitric Oxide Sensing through 1,2,3,4-Oxatriazole
Formation from Acylhydrazide: A Kinetic Study
Abu Saleh Musha Islam, Rahul Bhowmick, Bidhan Chandra Garain, Atul Katarkar, and Mahammad Ali
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02110 • Publication Date (Web): 08 Oct 2018
Downloaded from http://pubs.acs.org on October 8, 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 11
The Journal of Organic Chemistry
1
2
3
4
5
6
Nitric Oxide Sensing through 1,2,3,4-Oxatriazole Formation
from Acylhydrazide: A Kinetic Study
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
Abu Saleh Musha Islam , Rahul Bhowmick , Bidhan Chandra Garain , Atul Katarkar , and Ma-
hammad Ali
*†
†
Department of Chemistry, Jadavpur University, 188 Raja S.C. Mallick Road, Kolkata 700 032, India.
E-mail: m_ali2062@yahoo.com, mali@chemistry.jdvu.ac.in
Department of Biochemistry, University of Lausanne, Ch. des Boveresses 155, 1066 Epalinges, Switzerland.
‡
ABSTRACT: A simple molecular probe displays highly selective turn-on response towards NO by the unprecedented NO-
induced formation of 1,2,3,4-oxatriazole ring exhibiting no interference from various endogenous biomolecules including
DHA, AA etc. Kinetics of the reactions between NO and the probe provide a mechanistic insight into the formation of
1
,2,3,4-oxatriazole which showed that though initially 1,2,3,4-oxatriazole is formed and extractable in solid form, it exists
in equilibrium with ring opened azide form which ultimately hydrolysed and converted to carboxylic acid and nitrate. The
reaction displays second-order dependence on [NO] and first-order on [Probe]. The probe is water soluble, cell permeable
and non-cytotoxic and appropriates for live cell imaging. This constitutes the first report where, there is a direct evidence
of NO-induced ring closing reaction of acyl hydrazide moiety leading to the formation of 1,2,3,4-oxatriazole.
1
6
INTRODUCTION
er is oxidative deamination process. However, it exhibits
some unexpected limitations which include: (a) oxidation
of aromatic diamines and (b) reactions with other reactive
oxygen/nitrogen species. All these may result with the
Nitric oxide (NO) is an extra- and intracellular messen-
ger molecule mediating diverse signaling pathways in
target cells. It plays important roles in many physiological
processes that include neuronal signaling, inflammatory
response, modulation of ion channels, immune response,
phagocytic defense mechanism, penile erection, cardio-
vascular homeostasis and decompensation in atherogene-
1
6-24
generation of fluorescent species.
Another important
methods of NO sensing involve oxidative deamination of
aromatic amines, diazotization leading to ring formation
25-39
and N-nitrosation.
1
Very recently, we have synthesized a thiosemicarbazide
containing moiety for the selective detection of NO.
Where, thiosemicarbazide group reacts with NO/O to
form 1,3,4-oxadiazole and eliminates thionitrous acid.
Acylhydrazide, an important organic intermediate, is used
in the pharmaceutical industry for the synthesis of
nifuratrone. Protein and carbohydrate chemistry rou-
tinely utilizes hydrazide functionality. Hydrazides are
also used in the modification of oligonucleotides.
Herein, we have developed a new acylhydrazide based
molecular probes for NO detection in an oxygenated
aqueous medium leading to the formation of 1,2,3,4-
oxatriazole ring without any interference by ascorbic acid
AA), dehydroascorbic acid (DHA) or other reactive ni-
trogen (RNS) and Oxygen (ROS) species. Previously, it
sis. Depending upon the location and concentration, NO
plays major roles in cardiovascular system and central and
peripheral nervous systems. In contrast, micro molar
concentrations of NO not only forms the reactive nitro-
gen species (RNS) leading to neurodegenerative disorders
and carcinogenesis, but also provides a defense against
2
2
40
41
3
,4
invading pathogens.
NO, a diatomic free radical, un-
42
dergoes a variety of reactions with other radicals in tis-
sues, generating highly reactive nitrosating species in-
volved in nitrosation or oxidation of zinc finger-
43
5
containing proteins in DNA repair.
NO being lipid-soluble, highly reactive and easily dif-
fuseable in cells and tissues makes it difficult to capture
and detect. Several methods, like, chemiluminecence,
electrochemical, fluorescence and EPR etc. have been
proved to be useful for the in vitro and in vivo analysis of
(
44,45
was reported
that 1,2,3,4-oxatriazole moiety may be
6-15
formed from acyl hydrazide by the reaction with NaNO2
in strongly acidic medium; however, this constitutes the
first report where a direct reaction between NO and acyl
hydrazide leads to 1,2,3,4-oxatriazole in quantitative yield
under the physiological pH conditions at very low (micro
molar range) concentration. Detailed kinetic studies and
product analysis shed some light on the mechanism of
such reaction. Moreover, aqueous solubility makes it a
NO. In view of sensitivity, selectivity, spatiotemporal
resolution, fluorescence technique is considered as the
most favorable method for the detection of endogenous
NO. There are a number of inorganic and small organic
fluorescent NO probes, many of which are involved in the
reaction of aromatic diamines with NO/O forming an
electron deficient triazole moiety, that modules the PET
2
(
photo induced electron transfer) mechanism and anoth-
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 11
potent molecular probe for in vitro application for moni-
Spectral Response of QAH toward NO. The K (ap-
f
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
toring nitric oxide.
parent formation constant) for the reaction between QAH
and NO was calculated by the fluorescence titration
method maintaining fixed QAH concentration at 20 μM
and varied the NO concentrations between 0 - 40.0 μM in
aqueous HEPES buffer at 25 °C. A regular enhancement of
an emission peak at ~490 nm was observed with the in-
cremental addition of NO leading to a 23-fold fluores-
cence enhancement with λex = 380 nm (Figure 1). We
have also performed the excitation wavelength variation
study, where approximately fourteen and seven fold of
fluorescence enhancements were observed when QAH
was excited at 400 and 410 nm respectively, in the pres-
ence of excess NO. It is sufficiently high for the detection
of NO and also for the intracellular applications (Figure
S5).
RESULTS AND DISCUSSION
The receptor (quinolin-8-yloxy)-acetic acid hydrazide
(
(
QAH) was synthesized in two steps. In the initial step
quinolin-8-yloxy)-acetic acid ethyl ester was synthesized
from a reaction between 8-hydroxyquinoline and ethyl
bromoacetate in 1:1 mole ratio in dry acetone in the pres-
ence of anhydrous K CO . In the second step, ester was
2
3
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
reacted with hydrazine hydrate under reflux to afford
white coloured QAH (Scheme 1). The pure material was
1
13
1
characterized by H-NMR (Figure S1), C{ H}-NMR (Fig-
ure S2), HRMS (Figure S3) and FTIR (Figure S4) studies.
Scheme 1. Synthetic route of QAH
An excellent linear curve was obtained on plotting FI as
a function of [NO] up to 40 µM. On further addition of
NO the FI remains almost unaltered. The linear part was
46
analyzed using eqn. 1.
ꢂꢃꢄꢅꢆꢅꢇꢈ
ꢉꢃꢆꢅꢇꢈ
ꢀ
ꢁ
(1)
Where a and b are FIs of the free probe and in the pres-
ence of excess NO (≥ 40 µM), c = K . Assuming 1>> c*x
f
The QAH in aerated aqueous buffer was found to dis-
play a highly sensitive and selective fluorogenic response
towards NO. The idea of inclusion of 8-hydroxyquinoline
as fluorophore unit comes from the fact that it is an im-
portant moiety in many biological activities.
and n = 1 eqn. 1. turns to y= a+b*c*x. The b*c corresponds
to the slope of the curve, which ultimately gives c = K =
f
4
-1
(
3.01 ± 0.15) x 10 M .
Mechanism of fluorescence response of receptor
QAH to NO. The PET-ON effect from the high electron
rich hydrazide group to the quinoline fluorophore makes
parent compound QAH very weakly fluorescent. Howev-
er, due to the formation of electron deficient 1,2,3,4-
oxatriazole moiety out of the reaction between QAH and
NO in aerated water, the PET process is blocked.
(
Scheme 2) (vide infra). As a result, a remarkable turn-on
fluorescence response is observed at λex = 380 nm (Figure
).
1
Scheme 2. Schematic presentation of the PET effect
towards Fluorescence response of QAH on interac-
tion with NO/O2.
Figure 1. Fluorescence spectra of QAH (20 μM) upon reac-
tion with various amounts of NO (0-40 μM) in 10 mM HEPES
buffer (pH 7.20, µ = 0.10 M NaCl at 25 °C, λex = 380 nm). Inset
is the plot of FI vs. [NO] (S.D. = ±3).
To shed some light on the mechanism of fluorescence
enhancement for the reaction between QAH and NO we
have isolated the product of the reaction and character-
ized by exploiting different spectroscopic techniques.
Here, QAH (5 mmole) was reacted with nitric oxide in
acetonitrile in open air and the light yellow product was
isolated and purified by recrystallization from ethanol
and elucidated by NMR (Figures S6 and S7) (in DMSO-
d ), HRMS (Figure S8) and FTIR (Figure S9) spectrome-
6
+
try. The ESI-MS
peak at 248.0983 (1,2,3,4-
ACS Paragon Plus Environment

Page 3 of 11
The Journal of Organic Chemistry
+
oxatriazole+H O ) clearly suggest the formation of 1,2,3,4-
do-first-order rate constants) were extracted by the grafit
program.
When we plot kobs vs. [NO] it yields a non-linear curve
3
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
oxatriazole (QOT). The receptor QAH showed IR absorp-
tion bands at 3321 cm , 3136 cm and 1660 cm corre-
-
1
-1
-1
sponding to -NH , -NH and -C=O stretching vibrations,
with an upward curvature. However, when we plot k as
a function of [NO] it gives an excellent straight line indi-
2
obs
2
respectively. The product (QOT) showed -C=N and -N=N
–
1
–1
frequencies at 1679 cm and 1646 cm respectively de-
cating [NO] dependent second-order kinetics (Figure 3)
which was further confirmed by a plot of log(kobs) vs.
log[NO] yielding a slope = (2.2± 0.03) (Figure S14) with R
= 0.999. In a second experiment, we have determined the
dependence of rate on [QAH]. Here the [QAH] was varied
ranging between 20–100 μM w.r.t fixed [NO] i.e. 5 μM. A
plot of logkobs vs. log[QAH] gives slope (1.00 ± 0.26) (Fig-
ure S15) with R = 0.997, which also clearly indicates
[QAH] dependent first-order kinetics.
voiding any peaks for -NH , -NH and -C=O groups. Thus,
2
it clearly demonstrates that on reaction with NO, IR
peaks corresponding to -C=O in QAH is replaced by new
–
1
-1
peaks for -C=N and -N=N at 1679 cm and 1646 cm
4
4,45
(
sym.) respectively in QOT
(Figure S9) signifying the
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
3
1
formation of 1,2,3,4-oxatriazole. The C{ H}-NMR studies
also support this fact showing a shift in -C=O and -OCH2
(
167.3 and 68.7 ppm) signals of QAH to 170.1 and 66.2
ppm for -C=N and -OCH carbon atom in the QOT. Also
2
1
from the H-NMR titration in CD CN it showed that with
3
increasing the concentration of NO the peak correspond-
ing to –NH (4.04 ppm) and –NH (9.38 ppm) of the probe
2
(
QAH) gradually diminishes (Figure S10) and ultimately
vanishes to the base line. We run another IR spectrum
after few days of isolation of the product QOT; a very
-1
small peak at 2073 cm is observed due to the opening of
the oxatriazole ring and formation of a very small quanti-
ty of azide product (Figure S11). We attempted to grow
single crystals of QOT in CH CN and after one month
3
single crystals of the hydrolysis product of QOT was
formed which was characterized by single crystal X-ray
diffraction studies (Figure 2). So with time QOT is con-
verted to carboxylic acid very slowly through the inter-
47
mediate azide form (Scheme 3). We also studied the
stability of the probe (QAH) in CD CN and D O (1:99 v/v)
3
2
1
by H-NMR study. After 24 hrs. no observable change in
H-NMR spectra (Figure S12) was noticed. So the sensor
1
QAH is stable in pure water, at least for 24 h.
2
Figure 3. Plots of kobs vs. [NO] (Blue) and [NO] (Red) rep-
resents the reaction among QAH and NO. Conditions are:
[
QAH] = 5 μM and [NO] = (30 to 170) μM, 10 mM HEPES
buffer (pH = 7.2 , NaCl = 0.10 M, temperature = 15 ºC) em-
ploying fluorimetric techniques (S.D. = ±3).
Tentative reaction sequences can be framed as:
ꢎ
ꢏ
2
ꢊꢋ ꢌ ꢋ → 2ꢊꢋ
ꢐ2ꢑ
ꢐ3ꢑ
ꢐ4ꢑ
(5)
(6)
ꢍ
ꢍ
ꢎ
ꢒ
ꢊꢋ ꢌ ꢊꢋ → ꢊ ꢋ
ꢊ ꢋ ꢌ ꢔ ꢋ → 2ꢔ ꢌ 2ꢊꢋꢖ
ꢍ
ꢍ
ꢓ
ꢎ
ꢕ
ꢃ
Figure 2. Molecular view of 8-Carboxymethoxy-
quinolinium ion.
ꢍ
ꢓ
ꢍ
ꢍ
ꢎ
ꢘ
ꢖ
ꢃ
ꢊ ꢋ ꢌ ꢗ → ꢗ ꢙ ꢊꢋ ꢌ ꢊꢋ ꢌ ꢔ
ꢗ ꢙ ꢊꢋ ꢌ ꢔꢃ ꢝꢞꢟ ꢗ ꢌ ꢔ
ꢍ ꢓ
ꢍ
ꢚꢂꢛꢜ
ꢠ
Fluorometric Kinetic Studies. Pseudo-first-order
conditions were maintained for the kinetic studies of the
reaction of QAH with aqueous NO at pH 7.2 and 15 °C by
keeping QAH at 5 μM and NO concentration was varied
in the range 30-170 μM (Figure 3). The time dependent
fluorescence response and corresponding growth curve
for [QAH] = 5.0 ꢀM and [NO] = 10 ꢀM at 15 °C is illustrat-
ed in Figure S13. The kinetic traces at 490 nm display
single exponential growth curves which clearly manifest a
first-order dependence of rate on [QAH]. The kobs (pseu-
ꢍ
ꢋ
+
In the eqn (3) N
O
2
is formed which act as a NO donor
3
and react with L (QAH) to form L-NO, which leads to the
formation of the ring-closed 1,2,3,4-oxatriazole moiety (L')
(Scheme 3). The reported rate constants for the above
6
-2 -1 48
9
-1 -1 49
reactions are: k
= 6.33 x 10 M s ,
k
= 1.1 x10 M s ,
k [H O] = 1.6 x 10 s , . It clearly indicates that small con-
3 2
1
2
3
-1 50
centration of NO
mediates.
and N O are present as reactive inter-
2 3
2
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 11
Scheme 3. The proposed mechanism of the reaction of QAH with NO/O2.
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
Employing steady-state approximation for the concentra-
Determination of LOD (Limit of Detection) for NO
and Quantum yields. The 3σ method was used to de-
51
tion of NO and N O eqn (7) can be derived for the for-
2
2
3
mation of QOT (Lʹ).
termine the LOD of NO which was become 45.4 nM (Fig-
ure S18). The quantum yields (Φ) of QAH and QOT are
0.0179 and 0.3759 respectively, with an aqueous acidic
solution of quinine sulphate as standard, indicating QAH
as an example of ideal chemosensor for NO. As in macro-
ꢡꢢꢗꢠꢣ
ꢁ
ꢥ ꢥ ꢢꢗꢣ
ꢉ
ꢦ
ꢍ
ꢢꢊꢋꢣ ꢢꢋ ꢣ
ꢍ ꢜ
ꢐ7ꢑ
ꢜ
ꢡꢤ
2ꢐꢥ ꢢꢔ ꢋꢣ ꢌ ꢥ ꢢꢗꢣ
ꢓ ꢍ ꢦ
52
Experimentally, we observed that the reaction rate is
second-order in [NO] and first-order on [L] (QAH) which
helps us to assume that k [H O] >>k [L] and eqn. (7)
phase cultures the typical concentrations of NO is in
micro- to nanomolar range; our determined LOD value
clearly suggests that the present probe could be success-
fully used for the measurement of NO in these cell types.
3
2
4
turns to (8).
ꢡꢢꢗꢠꢣ
ꢥ ꢥ ꢢꢗꢣ
ꢉ
ꢦ
ꢍ
ꢁ
ꢢꢊꢋꢣ ꢢꢋ ꢣ ꢐ8ꢑ
ꢜ ꢍ ꢜ
ꢡꢤ
2 ꢥ ꢢꢔ ꢋꢣ
ꢓ ꢍ
Adoption of pseudo-first-order conditions with L as a
minor component and in open air (the dissolved [O ] =
2
t
2.5 mM at 25 °C) reduces eqn. (8) to eqn (9), where kobs
=
{
d[L’]/dt}/[L].
ꢠ
ꢍ
ꢜ
ꢥꢧꢄꢛ ꢁ ꢥ ꢢꢊꢋꢣ
(9)
Where,
ꢥꢉ ꢥꢦ
ꢥꢠ ꢁ 2
ꢐ10ꢑ
ꢢꢋꢍꢣ
ꢜ
ꢥ ꢢꢔ ꢋꢣ
ꢓ
ꢍ
6
-2 -1
The k' = (2.13 × 0.03) x 10 M s at 15 °C was extracted
2
from a linear curve fitting of the plot of kobs vs. [NO] .
With the help of reported values of k , k [H O], and k′, the
1
3
2
5
−1 −1
value of k = 4.31 × 10 M s was calculated and revealed
4
same order of magnitude for the alike reactions between
5
−1 −1
N O and different thiols:, GSH (2.9 × 10 M s ), N-acetyl
Figure 4. (a) Bar plot and (b) spectral plot of fluorescence
responses for the probe QAH at 490 nm towards different
reactive species. Conditions: HEPES buffer (10 mM, pH 7.2.);
2
3
5
−1 −1
5
−1 −1
cystein (1.5 × 10 M s ), HSA (0.3 × 10 M s ), Cys (2.6 ×
5
−1 −1
5
−1 −1 51
10 M s ), BSA (0.06 × 10 M s ) and also to our previ-
40
n-
ous results
.
QAH = 20 μM, X = 50 μM; λex = 380 nm (S.D. = ±3).
pH-Study: To check the practical applicability of the
probe under physiological conditions the dependence of
fluorescence intensities of QAH and QOT, formed after
reaction with NO, were investigated at different pH. The
results showed that the probe (10 µM) is very weakly
emissive in the pH range 4.0-9.0. But, in the presence of
Selectivity studies. The high specificity of the probe
(QAH) toward NO was determined by detecting its fluo-
rescence signal to a variety of reactive species, biomole-
2
–
cules, ROS, RNS and other species (50 μM) such as O
–
–
–
,
H O , NO , •OH, NO , ONOO , TEMPO, HCHO, gluta-
2 2 3 2
thione (GSH), dehydroascorbic acid (DHA), ascorbic acid
AA) and HNO. Figure 4 clearly highlights that no de-
20 µM of NO the FI was found to be significantly high
(
compared to QAH in the range pH 4.0-8.0 and then FI
abruptly decreases with pH (Figure S19). This observa-
tion clearly indicates that the FI of QOT remains suffi-
ciently high at pH~7.0, implying the safe use of QAH for
monitoring NO under physiological conditions.
tectable fluorescence signal of these reagents towards
QAH were observed under the identical reaction condi-
tions. Various inorganic anions (Figures S16(a) and
S16(b)) and cations (Figures S17(a) and S17(b)) also do
not interfere with the detection of NO
.
ACS Paragon Plus Environment

Page 5 of 11
The Journal of Organic Chemistry
Probing of PET process by DFT calculations: To con-
Both the cell lines exhibited good tolerability, which con-
strues QAH as a biocompatible NO sensor in live cells.
Before proceeds with fluorescence imaging, A549 and
Raw 264.7 cells were evaluated for exogenously and en-
dogenously release of NO by Griess assay (Figure 6b-c).
Endogenously induced NO sensing of QAH was evalu-
ated in Raw 264.7 murin macrophage cells. The cells were
co-stimulated with LPS (1.0 mg/mL) and IFN-γ (1000
U/mL) for 2, 4 and 6h followed by QAH (5 µM). Stimulat-
ed cells show bright blue fluorescence on treatment with
QAH compared to non-stimulated cells (Figure 7). The
bright blue fluorescence is due to endogenous NO and
was confirmed by further incubation of Raw 264.7 cells
with NO scavenger PTIO (200 mM) for 2h and then treat-
ed with QAH (5 µM) for 30 min, which showed a marked
covered up of blue fluorescence, implying that the intra-
cellular fluorescence changes are unquestionably due to
NO generation (Figure 7). Likely, bright blue fluores-
cence was seen on exogenously induced NO from DEA-
NONOate in the A549 cells (Figure 8). The fluorescence
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
firm the PET process we have carried out the theoretical
calculations on QAH and QOT using density functional
theory (DFT). The ground state optimized geometries
QAH and QOT are found to be stable without any imagi-
nary frequency. The QAH comprises of donor (hydrazide
group) and acceptor (quinoline) groups while QOT con-
sists of donor (1,2,3,4- oxatriazole group) and fluorophore
53-56
(quinoline moiety)
as based on the orbital distribution
diagram. So from the optimized ground state geometries
of QAH and QOT, we picked up the orbitals in which the
electron distributions are mainly located in the fluoro-
phore (acceptor) part. Similarly, we picked up the orbitals
in which the electron distributions are located in orbitals
of donor part (Figure 5). From Figure 5(a) for QAH the
HOMO energy of donor (hydrazide moiety) is found to be
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
-8.72 eV, whereas, in the case of fluorophore the HOMO
and LUMO energies are -8.90 eV and -0.64 eV, respective-
ly. As a result, HOMO energy of donor (hydrazide moie-
ty) (-8.72 eV) is higher than HOMO energy of the fluoro-
phore, therefore PET process can easily be happened. But
in QOT, formed after the reaction of QAH with NO, the
HOMO energy level of the donor is lower (-10.92 eV) than
HOMO energy of fluorophore (-8.77 eV) (Figure 5(b).
Thus, the PET process is blocked, which results in signifi-
cant enhancement of the fluorescence intensity.
intensity due to turn-on signal (QAH+NO) were quanti-
f
fied in endogenously and exogenously stimulated NO
(Figure 9 a and b). Eventually, we have also investigated
the detrimental toxic effect of QOT, the product of the
reaction between QAH and NO, on the cell viability.
Figure 6. (a) Graphical presentation of assay for Cell via-
bility. (b) Graphical representation (Mean
± SD) of
exogenous NO level measurement by Griess assay after add-
ing DEA NONOate 5 and 10 µM. (c) Graphical representation
(Mean ± SD) of endogenous measurement of NO level by
Griess assay after NO inducer treatment along with iNOS
inhibitor PTIO compared to control after 6 h.
Figure 5. Geometries and orbits of (a) QAH and (b) QOT
derived by DFT calculation. (DFT in CAM-B3LYP/6-31+G (d)
level).
The time-dependent cell viability of QOT generated
from the reaction between QAH + DEA-NONOate donor
(
5 and 10 μM) on A549 cells and -/+ stimulated Raw 264.7
Fluorescence cell imaging of NO (Exogenous and
Endogenous) in Live Cells. The cytotoxicity of the QAH
was evaluated in A549 and Raw 264.7 cells (Figure 6a).
cells up to 12 h does not exhibit any significant change in
time-dependent toxicity (Figure S20). All these observa-
ACS Paragon Plus Environment

The Journal of Organic Chemistry
tions point out the excellent biocompatibility and aptness
Page 6 of 11
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
of QAH for the recognition of NO in live cells by fluores-
cence technique.
Figure 9. (a) Quantified fluorescence (Mean ± SD) turn-on
in A549 cells after treatment with NO donor DEA-NONOate
in presence of QAH (b) Quantified fluorescence (Mean ± SD)
turn-on in Raw 264.7 cells after stimulation with LPS (1.0
mg/mL) and IFN-γ (1000 U/mL) for 2h, 4h and 6h in pres-
ence of QAH (5 µM) with or without iNOS inhibitor PTIO
compared to control. (a.u.) = Arbitrary unit.
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
CONCLUSION
In summary, we have successfully developed a simple
acyl hydrazide based fluorescent probe (QAH) for NO
sensing in purely aqueous medium. It detects exogenous
NO in A549 cells and endogenously generated NO in
RAW 264.7 cells. Moreover, the low cytotoxicity, high
selectivity, water solubility and excellent detection limit
of QAH probe towards NO make it a potential bio
imaging probe for in vitro monitoring of NO. To the best
of our knowledge, this is the first fluorescent probe
(QAH) for the detection of NO, through a novel mecha-
nism that leads to the formation of 1,2,3,4-oxatriazole het-
erocyclic moiety. Table S1 has been prepared for high-
lighting of LOD, reaction mechanism and kinetic studies
of some published NO probe. The kinetic studies of the
reactions between the probe and NO provide a mechanis-
tic insight into the formation of 1,2,3,4-oxatriazole which
showed that though initially 1,2,3,4-oxatriazole formed
and extractable in solid form, it exists in equilibrium with
its ring opened azide form which ultimately hydrolyzed
and converted to carboxylic acid and nitrate.
Figure 7. Fluorescence cell image of Raw 264.7 macro-
phage cells stimulated with LPS (1.0 mg/mL) + IFN-γ (1000
U/mL) with or without iNOS inhibitor PTIO (200 mM) for
2h, 4h and 6h followed by QAH (5 ꢀM) for 30 min. Intracel-
lular blue fluorescence was observed in response to NO in-
teraction with QAH and all the Images were captured at 40X
objective.
EXPERIMENTAL PROCEDURES
Materials and Methods: The reagent grade starting
materials such as Hydrazine hydrate, Ethyl-bromoacetate,
+
+
8-Hydroxyquinoline (Sigma Aldrich), salts of K , Na ,
2
+
2+
3+
3+
2+
2+
3+
2+
2+
2+
Ca , Mg , Cr , Al , Fe , Mn , Fe , Cu , Co , Ni ,
2
+
2+
2+
2+
Hg , Zn , Pb , Cd etc. and sodium salts of anions like
-
–
–
–
–
–
–
-
-
-
–
SCN , NO , NO , N , I , Br , F , BrO , HPO , IO , OAc ,
S2O3 , S O4 , PPi, SCN , NCS etc. with other reactive
species like H O , •OH, O ,TEMPO, NO , NO , ONOO
3
2
3
3
4
3
2–
2–
-
-
2
2–
–
–
–
2
2
3
2
,
GSH, ascorbic acid (AA), dehydroascorbic acid (DHA),
and DEA-NONOate (sodium salt) were purchased from
Sigma Aldrich and used without further purification. Sol-
vents like absolute ethanol, MeOH, CH CN etc. (Merck,
3
India) were of reagent grade and dried before use.
Figure 8. Fluorescence imaging of A549 cells incubated
with DEA-NONOate (5 and 10 µM) and QAH (5 and 10 µM).
Intracellular blue fluorescence was observed in response NO
interaction with QAH. Images were taken at 40X objective.
Physical Measurements: FTIR (Fourier Transform In-
-
1
frared Spectra) (4000 – 400 cm ) of the ligands (QAH)
and product (QOT) of the reaction with NO were record-
ed on a Perkin-Elmer RX I FTIR spectrophotometer on
ACS Paragon Plus Environment

Page 7 of 11
The Journal of Organic Chemistry
solid KBr discs. UV-Vis spectra were recorded on an Ag-
ꢋꢬꢛꢜꢭ ꢅ ꢮꢛꢂꢨꢩꢪꢫ
Фꢛꢨꢂꢩꢪꢫ
ꢁ
ꢅ Фꢛꢜꢭ
ꢐ12ꢑ
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
ilent 8453 Diode-array spectrophotometer. PTI (Model
QM-40) spectrofluorimeter was used for fluorescence
ꢋꢬꢯꢂꢨꢩꢪꢫ ꢅ ꢮꢛꢜꢭ
1
studies. Bruker 300 MHz spectrometer was used to run H
Where, the respective areas of the sample and standard
under the fluorescence spectral curves are represented by
Asample and A . The corresponding optical densities of the
1
3
1
and C{ H}-NMR spectra in CD CN and DMSO-d using
3
6
+
trimethylsilane (δ = 0) as an internal standard. ESI-MS
std
(m/z) of the probe (hereafter designated as QAH) and the
reaction product (hereafter designated as QOT) were rec-
orded on a HRMS spectrometer (Model: QTof Micro
YA263).
standard and sample at λ are given by OD and ODsample
ex
std
respectively. Aqueous acidic solution of quininesulfate
was used as the standard with Фstd = 0.54.
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
Computational method: To establish the PET mecha-
nism, the geometry of QAH and QOT were optimized by
CAM-B3LYP function using 6-31+G(d) basis set. On the
basis of all optimized ground state structures of QAH and
QOT, the absorption spectra were calculated by TD-DFT
time-dependent) method applying CAM-B3LYP/6-
1+G(d) basis set. All calculations of the molecules were
carried out using the Gaussian 09 program package and
also PCM (polarizable continuum model) was adopted for
-
3
Preparation of Stock Solutions: 10 ml of 1.0 x 10 M
stock solution of the probe (QAH) was prepared by dis-
solving it in two drops of CH CN (~1%) and then volume
3
was adjusted with H O (HPLC grade). Nitric oxide gas,
2
57
was purified by passing through solid NaOH pellets, and
then purzed through deoxygenated deionized water for 15
min in a sealed vial, which results in NO concentration of
(
3
6
0
-
3
1
.74 x 10 M. The reported method was used to prepare
-
58
solutions of •OH and ONOO . Angeli's salt was used a
source of HNO. The stock solutions (1.0 x 10 M) of dif-
ferent cations, anions and different molecular species
solvent (H O) effects.
2
59
-3
Fluorescence Imaging Experiments:
were prepared in H O. All the experiments were carried
2
out in 10.0 mM HEPES buffer at pH 7.2 and ionic strength
was maintained at 0.10 M (NaCl). 2.5 ml of the 10.0 mM
HEPES buffer was pipetted out into a cuvette and 50 µL of
Cell viability assay: The lung carcinoma A549 cells and
Raw 264.7 murine macrophages, grown in Dulbecco's
modified Eagle's (DMEM) medium were supplemented
-3
the probe (1.0 x 10 M) was added, which gives the probe
with 10% FBS and 1% antibiotic at 37 °C with 5% CO . Cell
2
concentration 20 ꢀM, and then variable concentration (0
viability of the ligand QAH was evaluated against A549
5
-
40 µM) of NO was added incrementally and fluores-
and Raw 264.7 cells. Briefly, 1×10 cells were seeded in 96-
cence spectra were recorded for each solution.
well culture plate and allowed to settle down for over-
night at 37°C. Cells were then treated with ligands QAH
with gradual increasing concentration (ranging 10-100
µM) for 24 h. 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) solution (10 mg/ml
Kinetic Studies: Kinetic studies were carried out under
pseudo-first-order conditions with QAH as a minor com-
ponent (5 μM) and nitric oxide concentration was varied
between 30 and 170 μM at pH 7.20, at 15 °C. The depend-
ence of rate on [QAH] was also determined keeping NO
as a minor component (5 μM) and [QAH] was varied in
the range 20–100 μM. The first-order dependence of rate
on [QAH] was confirmed by a plot of logkobs vs. log[QAH]
giving a slope (1.00 ± 0.26) with R = 0.999. Similarly a se-
cond order dependence on [NO] was confirmed by a plot
of logkobs vs. log[NO] with slope (2.2± 0.03) with R =
1
xPBS) was supplemented to each 96-well and incubated
at 37°C for 3 h. Media were removed from all wells and 100
μl acidic isopropyl alcohol was added. The intracellular
formazan crystals (blue-violet) were solubilized in 0.04 N
acidic isopropyl alcohol and absorbance was measured at
5
95 nm with a micro plate reader (EMax Precision Micro-
Plate Reader, Molecular Devices, USA). Experiments were
repeated three times and relative cell viability was ex-
pressed as a percentage (%) compared to untreated con-
trol cells. Endogenous and exogenous NO generation was
0
.999.
61
Calculation of LOD: For the LOD (Limit of Detection)
measured by Griess assay .
calculation of NO 3σ method was used (eqn. 11).
LOD = 3 x S /S (11)
Where, the standard deviation of the intercept of the
In-Vitro Cell incubation and imaging: A549 and Raw
264.7 murine macrophages cells were taken for exogenous
and endogenous NO detection respectively. The cells
were cultured on glass coverslip inside 35x10 mm culture
dishes. Exogenously, A549 cells were treated with sodium
salt of nitric oxide donor DEA-NONOate (5 µM and 10
d
blank is represented by S and is acquired from a plot of
d
fluorescence intensity (FI) vs. [QAH]. The slope of the
linear plot of FI vs. [NO] of the fluorescence titration data
is represented by S.
µM) and incubate at 37°C with 5% CO for 30 min and
2
then washed 3 times with 1x PBS followed by treatment
with QAH (5 and 10 µM) for 30 min, washed and live cell
imaging by fluorescence microscope (Carl Zeiss, Germa-
ny) was completed. Endogenously, Raw 264.7 cells were
co-stimulated with LPS (1.0 mg/mL) and IFN-γ (1000
U/mL) for 2, 4 and 6 h, and without co-stimulant (con-
Quantum Yield: Fluorescence quantum yields (Ф) of
QAH and QOT were determined with the help of eqn. 12
ACS Paragon Plus Environment

The Journal of Organic Chemistry
trol) and further incubated with QAH (5 µM) for 30 min.
Page 8 of 11
Financial
supports
from
DST
(Ref.
No.
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
809(Sanc)/ST/P/S&T/4G-9/2104) West Bengal and CSIR (Ref.
01(2896)/17/EMR-II), New Delhi, India are gratefully
acknowledged. Thanks to M. Dolai for helping in crystal data
solving.
The cellular fluorescence was accompanying only with
NO generation was confirm by NO scavenger PTIO (2-
Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide)
treatment. Raw 264.7 cells were co-stimulated with LPS
(1.0 mg/mL) and IFN-γ (1000 U/mL) for 2h, 4h and 6 h,
along with PTIO (200 mM) followed by treated with the
QAH (5 µM) for 30 min and live fluorescence images were
performed.
REFERENCES
(
1) O’Dell, T. J.; Hawkins, R. D.; Kandel, E. R. Arancio O. Tests
of the roles of two diffusible substances in long-term poten-
tiation: evidence for nitric oxide as a possible early retro-
grade messenger. Proc Natl. Acad. Sci. USA 1991, 88, 11285 –
Syntheses of (Quinolin-8-yloxy)-acetic acid hydra-
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
11289.
zide (QAH). QAH was synthised in two steps by the liter-
(2) Butler, A. R.; Williams, D. L. H. The physiological role of
nitric oxide. Chem. Soc. Rev. 1993, 22, 233–241.
(3) Moncada, S.; Palmer, R. M.; Higgs, E. A. Nitric oxide: phys-
iology, pathophysiology, and pharmacology Pharmacol.
Rev. 1991, 43, 109-142.;
62
ature method. A mixture of 8-hydroxyquinoline (0.06
mol, 8.71 g), ethyl bromoacetate (0.06 mol, 10.02 g) and
anhydrous K CO (0.06 mol, 8.29 g) in dry acetone was
2
3
refluxed for 17 h. to afford (quinolin-8-yloxy)-acetic acid
ethyl ester. The solid residue thereby formed was re-
moved by filtration and then the solvent was removed
under reduced pressure to get the desired product which
was purified by column chromatography on silica gel,
using ethyl acetate: petroleum ether (1:3) as the yellow oil.
Yield 11.79 g (84.6 %).
(
4) Conner, E. M.; Grisham, M. B. Nitric Oxide: Biochemistry,
Physiology, and Pathophysiology. Methods Enzymol. 1995,
7, 3-13.
(5) Sidorkina O.; Espey M. G.; Miranda K. M.; Wink D. A.; La-
val J. Inhibition of Poly(ADP-ribose) Polymerase (PARP) by
Nitric Oxide and Reactive Nitrogen Oxide Species. Free
Radical Biol. Med. 2003, 35, 1431–1438
(
6) Brune, B.; Dimmeler, S.; Vedia L, M.; Lapetina, E. G. Nitric
Oxide: A Signal for ADP-Ribosylation of Proteins. Life Sci.
1994, 54, 61– 70.
The resulting ester (0.02 mol, 4.62 g) was dissolved in
ethanol to which hydrazine hydrate (0.4 mol) in ethanol
was added and the resulting mixture was refluxed on a
water bath for 7 h. After cooling, the precipitated solid
was filtered and washed with cold water, dried and recrys-
(7) Benjamin, J. P.; Shin, J. H.; Schoenfisch, M. H. Electrochem-
ical nitric oxide sensors for physiological measurements.
Chem. Soc. Rev. 2010, 39, 1925– 1935.
(
8) Hu, W.; Boateng, D.; Kong, J.; Zhang, X. Advancement of
Fluorescent Methods for Detection of Nitric Oxide. Austin J.
Biosens & Bioelectron. 2015, 1, 1– 9.
tallized from ethanol (Scheme 1). Yield 3.26 g (75%).
1
[
C11H11N3O2]; Molecular Weight 217.0851. H-NMR (300
MHz DMSO-d ): 4.41 (s, 2H, -NH ), 4.75 (s, 2H, -CH ),
(9) Gabe Y.; Urano Y.; Kikuchi K.; Kojima H.; Nagano T. Highly
Sensitive Fluorescence Probes for Nitric Oxide Based on Bo-
ron Dipyrromethene Chromophore Rational Design of Po-
tentially Useful Bioimaging Fluorescence Probe. J. Am.
Chem. Soc. 2004, 126, 3357–3367.
6
2
2
7
8
.25 (d, 1H), 7.50 - 7.61 (m, 3H, –ArH), 8.38 (d, 1H,–ArH),
.92 (d, 1H, –ArH), 9.46 (s, 1H, -NH) (Figure S1). C{ H}-
1
3
1
NMR (in DMSO-d )(δ, ppm): 167.3, 154.3, 149.8, 140.2,
6
136.6, 129.6, 127.2, 122.5, 121.4, 112.1, 68.7. (Figure S2). ESI-
(
10) Sasaki, E.; Kojima H.; Nishimatsu H.; Urano Y.; Kikuchi K.;
Hirata Y.; Nagano T. Highly Sensitive Near-Infrared Fluo-
rescent Probes for Nitric Oxide and Their Application to
Isolated Organs. J. Am. Chem. Soc. 2005, 127, 3684–3685.
+
+
MS (m/z): (QAH+H ) 218.0958 found; calculated
218.0930. (Figure S3).
(11) Yang ,Y.; Seidlits, S. K.; Adams, M. M.; Lynch V. M.;
Schmidt, C. E.; Anslyn, E. V.; Shear, J. B. A Highly Selective
Low-Background Fluorescent Imaging Agent for Nitric Ox-
ide. J. Am. Chem. Soc. 2010, 132, 13114–14116.
ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
(
12) McQuade, L. E.; Pluth, M. D.; Lippard, S. J. Mechanism of
Nitric Oxide Reactivity and Fluorescence Enhancement of
the NO-Specific Probe CuFL1. Inorg. Chem. 2010, 49, 8025–
8033.
Fluorescence and Characterization data of compounds QAH
1
13
and QOT: H NMR, C-NMR, IR, HRMS, pH effect, DFT com-
putational data, Primary Kinetic data, X-ray crystallography
data, CIF file (PDF)
(13) McQuade, L. E.; Lippard, S. J. Fluorescence-Based Nitric
Oxide Sensing by Cu(II) Complexes That Can Be Trapped in
Living Cells. Inorg. Chem. 2010, 49, 7464–7471.
(
14) Lim, M. H.; Lippard, S. J. Fluorescence-Based Nitric Oxide
Detection by Ruthenium Porphyrin Fluorophore Complex-
es. Inorg. Chem. 2004, 43, 6366-6370.
AUTHOR INFORMATION
Corresponding Author
(15) Li, H.; Wan, A. Fluorescent Probes for Real-time
*
E-mail: m_ali2062@yahoo.com
Measurement of Nitric Oxide in Living Cells. Analyst 2015,
‡
Present Address
1
40, 7129– 7141.
ORCID
M. Ali: 0000-0003-0756-0468
(
16) Beltrán, A.; Burguete, M. I.; Abánades, D. R.; Pérez-Sala, S.
D.; Luis, V.; Galindo, F. Turn-on Fluorescent Probes for Ni-
tric Oxide Sensing Based on the Ortho-Hydroxyamino
Structure Showing no Interference with Dehydroascorbic
acid. Chem. Commun. 2014, 50, 3579–3581.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
ACS Paragon Plus Environment

Page 9 of 11
The Journal of Organic Chemistry
(
17) Lim, M. H.; Lippard, S. J. Copper Complexes for Fluores-
fluorescent probes for imaging the biological signaling mol-
ecule nitric oxide in living cells and organisms. J. Mater.
Chem. B. 2017, 5, 2483−2490.
1
2
3
4
5
6
7
8
cence-Based NO Detection in Aqueous Solution. J. Am.
Chem. Soc. 2005, 127, 12170-12171.
(
18) Lim, M. H.; Lippard, S. J. Fluorescent Nitric Oxide Detec-
tion by Copper Complexes Bearing Anthracenyl and Dansyl
Fluorophore Ligands. Inorg. Chem. 2006, 45, 8980-8989.
19) Sun, C.; Shi, W.; Song, Y.; Chen, W.; Ma, H. An unprece-
dented strategy for selective and sensitive fluorescence de-
tection of nitric oxide based on its reaction with a selenide.
Chem. Commun. 2011, 47, 8638–8640.
(35) Reinhardt, C. J.; Zhou, E. Y.; Jorgensen, M. D.; Partipilo, G.; .
Chan, J. A Ratiometric Acoustogenic Probe for in Vivo Im-
aging of Endogenous Nitric Oxide. J. Am. Chem. Soc. 2018,
140 , 1011–1018.
(36) Mao, Z.; Jiang, H.; Song, X.; Hu, W.; Liu, Z. Development of
a Silicon-Rhodamine Based Near-Infrared Emissive Two-
Photon Fluorescent Probe for Nitric Oxide. Anal. Chem.
2017, 89, 9620−9624.
(37) Mia, J.; Huo, Y.; Lv, X.; Li, Z.; Cao, H.; Shi, H.; Shi, Y.; Guo,
W. Fast-response and highly selective fluorescent probes for
biological signaling molecule NO based on N-nitrosation of
electron-rich aromatic secondary amines. Biomaterials,
2016, 78, 11-19.
(38) Dai, C. G.; Wang, J. L.; Fu,Y. L.; Zhou, H. P.; Song, Q. H.
Selective and Real-Time Detection of Nitric Oxide by a
Two-Photon Fluorescent Probe in Live Cells and Tissue
Slices. Anal. Chem., 2017, 89, 10511−10519.
(
(
20) Smith, R. C.; Tennyson, A. G.; Lim, M. H.; Lippard S. J. Con-
jugated Polymer-Based Fluorescence Turn-On Sensor for
Nitric Oxide. Org. Lett. 2005, 7, 3573-3575.
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
(21) Shiue, T. W.; Chen, Y. H.; Wu, C. M.; Singh, G.; Chen, H. Y.;
Hung, C. H.; Liaw, W. F.; Wang, Y. M. Nitric Oxide Turn-on
Fluorescent Probe Based on Deamination of Aromatic Pri-
mary Monoamines. Inorg. Chem. 2012, 51, 5400−5408.
(22) Kojima, H.; Hirotani, M.; Nakatsubo, N.; Kikuchi, K.; Ura-
no, Y.; Higuchi, T.; Hirata, Y.; Nagano, T. Bioimaging of Ni-
tric Oxide with Fluorescent Indicators Based on the Rho-
damine Chromophore. Anal. Chem. 2001, 73, 1967-1973.
(23) Yu, H.; Zhang, X.; Xiao, Y.; Zou, W.; Wang, L.; Jin, L. Tar-
getable Fluorescent Probe for Monitoring Exogenous and
Endogenous NO in Mitochondria of Living Cells. Anal.
Chem. 2013, 85, 7076−7084.
(39) Muthuraj, B.; Deshmukh, R.; Trivedi, V.; Iyer, P. K. Highly
2+
Selective Probe Detects Cu and Endogenous NO Gas in
Living Cell. ACS Appl. Mater. Interfaces, 2014, 6, 6562−6569.
(40) Islam, A S M.; Bhowmick, R.; Pal, K.; Katarkar, A.;
Chaudhuri, K.; Ali, M. A Smart Molecule for Selective Sens-
ing of Nitric Oxide: Conversion of NO to HSNO; Relevance
of Biological HSNO Formation. Inorg. Chem. 2017, 56, 4324-
4331.
(
24) Dong, X.; Heo, C. H.; Chen, S.; Kim, H. M.; Liu, Z. Quino-
line-Based Two-Photon Fluorescent Probe for Nitric Oxide
in Live Cells and Tissues. Anal. Chem. 2014, 86, 308−311.
25) Yao, H. W.; Zhu, X. Y.; Guo, X. F.; Wang, H. An Amphiphilic
Fluorescent Probe Designed for Extracellular Visualization
of Nitric Oxide Released from Living Cells. Anal. Chem.
2016, 88, 9014-9021.
(
(41) Marion, L.; Lavigne, R.; Lemay, L. The Stracture of
Sedamin. Can. J. Chem. 1951, 29, 843–847.
(42) Raddatz, S.; Mueller-Ibeler, J.; Kluge, J. L.; Burdinski, G.;
Havens, J. R.; Onofrey, T. J.; Wang, D.; Schweitzera,
Markus. Hydrazide oligonucleotides: new chemical modifi-
cation for chip array attachment and conjugation. Nucleic
Acid Res. 2002, 21, 4793–4802.
(43) de Lumley-Woodyear, T.; Campbell ,C. N.; Heller, A. Direct
Enzyme-Amplified Electrical Recognition of a 30-Base Mod-
el Oligonucleotide. J. Am. Chem. Soc. 1996, 118, 5504–5505.
(44) Ibrahim, M. K.; Adl, K. E.; Zayed, M. F.; Mahdy, H. A. De-
(
(
(
(
26) Dai, Z.; Tian, L.; Song, B.; Liu, X.; Yuan, J. Development of a
novel lysosome-targetable timegated luminescence probe
for ratiometric and luminescence lifetime detection of nitric
oxide in vivo. Chem Sci. 2017, 8, 1969–1976.
27) Wang, C.; Song, X.; Han, Z.; Li, X.; Xu, Y.; Xiao, Y. Monitor-
ing Nitric Oxide in Subcellular Compartments by Hybrid
Probe Based on Rhodamine Spirolactam and SNAP-tag.
ACS Chem. Biol. 2016, 11, 2033-2040.
sign, synthesis, docking, and biological evaluation of some
novel 5-chloro-2-substituted sulfanylbenzoxazole deriva-
tives as anticonvulsant agents. Med. Chem. Res. 2015, 24,
99–114.
28) Mao, Z.; Feng, W.; Li, Z.; Zeng, L.; Lv, W.; Liu, Z. NIR in, far
red out: developing a two-photon fluorescent probe for
tracking nitric oxide in deep tissue. Chem. Sci. 2016, 7, 5230-
5
235.
(45) Azab, M. E.; Madkour,H. M. F.; Ibraheem, M. A. E. The
Utility of 2-(5,6,7,8-Tetrahydrobenzo[b]thieno- [2,3-
d]pyrimidin-4-yloxy) Acethydrazide in Heterocyclic Syn-
thesis. Phosphorus, Sulfur and Silicon 2006, 181, 1299–1313.
(46) Islam, A. S. M.; Alam, R.; Katarkar, A.; Chaudhuri, K.; Ali,
M. Di-oxime Based Selective Fluorescent Probe for Arse-
nate and Arsenite Ions in a Purely Aqueous Medium with
Living Cell Imaging Applications and H-bonding Induced
Microstructure Formation. Analyst 2015, 140, 2979-2983.
(47) Wu, C. M.; Chen, Y. H.; Dayananda, K.; Shiue, T. W.; Hung,
C. H.; Liaw, W. F.; Chen, P. Y.; Wang, Y. M. Sensitivity
evaluation of rhodamine B hydrazide towards nitric oxide
and its application for macrophage cells imaging. Anal.
Chem. Acta. 2011, 708, 141–148.
29) Tian, L.; Dai, Z.; Liu, X.; Song, B.; Ye, Z.; Yuan, J. Ratiometric
Time-Gated Luminescence Probe for Nitric Oxide Based on
an Apoferritin-Assembled Lanthanide Complex-Rhodamine
Luminescence Resonance Energy Transfer System. Anal.
Chem. 2015, 87, 10878−10885.
(
30) Tang, J.; Guo, Z.; Zhang, Y.; Bai, B.; Zhu, W. H. Rational
design of a fast and selective near infrared fluorescent probe
for targeted monitoring of endogenous nitric oxide. Chem.
Commun. 2017, 53, 10520- 10523.
(31) Huo, Y.; Miao, J.; Han, L.; Li, Y.; Li, Z.; Shib, Y.; Guo, W.
Selective and sensitive visualization of endogenous nitric
oxide in living cells and animals by a Sirhodamine deoxylac-
tam-based near-infrared fluorescent probe. Chem. Sci. 2017,
8, 6857–6864.
(
32) Yu, H.; Zhang, X.; Xiao, Y.; Zou, W.; Wang, L.; Jin, L. Tar-
getable Fluorescent Probe for Monitoring Exogenous and
Endogenous NO in Mitochondria of Living Cells. Anal.
Chem. 2013, 85, 7076−7084.
(
48) Kharitonov, V. G.; Sundquist, A. R.; Sharma, V. S. Kinetics
of nitric oxide autoxidation in aqueous solution. J. Biol.
Chem. 1994, 8, 5881-5883.
(
33) Wang, N.; Yu, X.; Zhang, K.; Mirkin, C. A.; Li, J. Upconver-
sion Nanoprobes for the Ratiometric Luminescent Sensing
of Nitric Oxide. J. Am. Chem. Soc. 2017, 139, 12354−12357.
34) Huo, Y.; Miao, J.; Li, Y.; Shi, Y.; Shia, H.; Guo, W. Aromatic
primary monoamine-based fast-response and highly specific
(49) Gra¨tzel, M.; Taniguchi, S.; Henglein, A. Pulse radiolytic
investigation of the oxidation of NO and of the equilibrium
(
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 11
N2O3 ⇄ NO + NO2 in aqueous solution. Phys. Chem. 1970,
and D. J. Fox, Gaussian 09, (Revision A.1), Gaussian, Inc.,
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
74, 488–492.
Wallingford, CT, 2009.
(50) Licht, W. R.; Tannenbaum, S. R.; Deen, W. M. Use of ascor-
bic acid to inhibit nitrosation: kinetic and mass transfer
considerations for an in vitro system. Carcinogenesis 1988,
9, 365–372.
(61) Alam, R.; Islam, A. S. M.; Sasmal, M.; Katarkar, A.; Ali, M. A
rhodamine-based turn-on nitric oxide sensor in aqueous
medium with endogenous cell imaging: an unusual for-
mation of nitrosohydroxylamine. Org. Biomol. Chem. 2018,
16, 3910-3920.
(51) Kharitonov, V. G.; Sundquist, A. R.; Sharma, V. S. Kinetics
of Nitrosation of Thiols by Nitric Oxide in the Presence of
Oxygen. J. Biol. Chem. 1995, 270, 28158–28164.
(62) Islam, A. S. M.; Bhowmick, R.; Mohammad, H.; Katarkar,
A.;Chaudhuri, K.; Ali, M. A Novel 8-hydroxyquinoline-
pyrazole Based Highly Sensitive and Selective Al(III) Sensor
in a purely Aqueous Medium with Intracellular Applica-
tion: Experimental and Computational Studies. New J.
Chem. 2016, 40, 4710-4719.
(52) Chen, T.; Zamora, R.; Zuckerbraun, B.; Billiar, T. R. Role of
Nitric Oxide in Liver Injury. Curr Mol Med. 2003 , 3, 519-
526.
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
(53) Zhang,Y. J.; Yang, W. J.; Wang, C. K. Effect of fluorophore
on sensing NO for newly synthesized PET-based two-
photon fluorescent probes. Chemical Physics 2016, 468, 37–
43.
(54) Xu, Z.; Ren, A. M.; Wang, D.; Guo, J. F.; Feng, J. K.; Yu, X. A
theoretical investigation on two latest two-photon pH fluo-
rescent probes. Journal of Photochemistry and Photobiology
A: Chemistry 2014, 293, 50–56.
(55) Xu, Z.; Ren, A. M.; Guo, J. F.; Liu, X. T.; Huang, S.; Feng, J.
K. A Theoretical Investigation of Two Typical Two-Photon
pH Fluorescent Probes. Photochemistry and Photobiology
2013, 89, 300–309.
(56) Mao, Z.; Jiang, H.; Song,X.; Hu, W.; Liu, Z. Development of
a Silicon-Rhodamine Based Near-Infrared Emissive Two-
Photon Fluorescent Probe for Nitric Oxide. Anal. Chem.
2017, 89, 9620−9624.
(57) Mesároš, Š.; Grunfeld, S.; Mesárošová, A.; Bustin, D.; Ma-
linski, T. Determination of nitric oxide saturated (stock) so-
lution by chronoamperometry on a porphyrine microelec-
trode. Anal. Chim. Acta. 1997, 339, 265−270.
(58) Miyamoto, S.; Martinez, G. R.; Martins, A. P. B.; Medeiros,
M. H. G.; Di Mascio, P. Direct Evidence of Singlet Molecu-
lar Oxygen [O2 (1Δg)] Production in the Reaction of Linole-
ic Acid Hydroperoxide with Peroxynitrite. J. Am. Chem. Soc.
2003, 125, 4510−4517.
(59) Wrobel, A. T.; Johnstone, T. C.; Liang, A. D.; Lippard, S. J.;
Fuentes, P. R. A Fast and Selective Near-Infrared Fluores-
cent Sensor for Multicolor Imaging of Biological Nitroxyl
(HNO). J. Am. Chem. Soc. 2014, 136, 4697−4705.
(60) M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M.
A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Men-
nucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.
P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Son-
nenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Ha-
segawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H.
Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F. Ogli-
aro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N.
Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A.
Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N.
Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V.
Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Strat-
mann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W.
Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G.
A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D.
Daniels, O¨. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski
ACS Paragon Plus Environment

Page 11 of 11
The Journal of Organic Chemistry
TOC
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
ACS Paragon Plus Environment