Triggered emission for rapid detection of hydrogen sulfide chaperoned by large Stokes shift

Article abstract of DOI:10.1016/j.jphotochem.2018.11.011

Current manuscript demonstrates the development of a reaction-based “turn-on” green emissive molecular probe (P4) for rapid detection of H₂S in physiological condition. The probe was found to exhibit low limit of detection (1.19 μM), along with high selectivity, large Stokes shift and good quantum efficiency. The change in the optical behaviour of the probe was also theoretically scrutinized employing density functional theory calculations which stand in the strong agreement with the photophysical outcomes. Given the demand for large Stokes dyes for sensing applications mainly because of their special photophysical properties, and as the reported number is very limited, the present probe with 110 nm Stokes shift is a good addition in the area of large Stokes dyes for H₂S recognition in physiological conditions.

Full text of DOI:10.1016/j.jphotochem.2018.11.011

Accepted Manuscript
Title: Triggered emission for rapid detection of hydrogen
sulfide chaperoned by large Stokes shift
Authors: Bidisha Biswas, M. Venkateswarulu, Pankaj Gaur,
Yamini Sharma, Sougata Sinha, Subrata Ghosh
PII:
S1010-6030(18)31300-5
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.11.011
JPC 11583
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
5 September 2018
12 October 2018
7 November 2018
Please cite this article as: Biswas B, Venkateswarulu M, Gaur P, Sharma Y, Sinha S,
Ghosh S, Triggered emission for rapid detection of hydrogen sulfide chaperoned by
large Stokes shift, Journal of Photochemistry and amp; Photobiology, A: Chemistry
(2018), https://doi.org/10.1016/j.jphotochem.2018.11.011
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Triggered emission for rapid detection of hydrogen sulfide chaperoned by
large Stokes shift
a
a
a
a
b,
Bidisha Biswas, M. Venkateswarulu, Pankaj Gaur, Yamini Sharma, Sougata Sinha, * and
a,
Subrata Ghosh *
aSchool of Basic Sciences, Indian Institute of Technology Mandi, Mandi-175001, H.P, India
bDepartment of Chemistry, Nalanda College of Engineering, Chandi- 803108, Bihar, India
*Corresponding author: Tel. +91-1905-267069; Fax: +91-1905-237924 (S. Ghosh)
E-mail address: subrata@iitmandi.ac.in (S. Ghosh)sinhasougatachemistry@gmail.com (S.
Sinha)
Graphical Abstract
1

Highlights:





Reaction-based 'turn-on' fluorescent probe for optical detection of H S;
2
One step synthesis with good yield;
Guided by mega-Stokes shift;
Low limit of detection and limit of quantification;
High selectivity over biothiols, bio-relevant cations and anions;
Abstract
Current manuscript demonstrates the development of a reaction-based "turn-on" green
emissive molecular probe (P4) for rapid detection of H S in physiological condition. The
2
probe was found to exhibit low limit of detection (1.19 µM), along with high selectivity,
large Stokes shift andgood quantum efficiency. The change in the optical behaviour of the
probe was also theoretically scrutinized employing density functional theory calculations
which stand in the strong agreement with the photophysical outcomes. Given the demand for
large Stokes dyes for sensing applications mainly because of their special photophysical
properties, and as the reported number is very limited, the present probe with 110 nm Stokes
shift is a good addition in the area of large Stokes dyes for H
conditions.
2
S recognition in physiological
Keywords: Fluorescent probe, Hydrogen sulfide, large Stokes shift, Anthracene, Azide
reduction
2

1
. Introduction
The volatile organosulfur species are well identified for their characteristic obnoxious odour
and considered to be toxic to human health as well as to the environment [1-3]. Amongst
these, hydrogen sulfide is the most commonmalodorousgas and often found to be associated
with numerous biological/physiological activities [1-14]. Literature reports also evidenced the
close attachment of diseases such as Alzheimer, Down’s Syndrome, liver cirrhosis, diabetes,
2
pulmonary hypertension etc. with H S concentration [8, 13-16]. Hence, efforts are being
made to get better insights of its physio-pathological importance and in this context,
development of simple cost-effective tools for monitoring
environmental/biological sample is on urgent demand.
H
2
S
level in
S, such as
Even though, a number of techniques are currently existing for detection of H
2
colorimetric [11, 17, 18], metal induced sulfide precipitation [19], electrochemical [20-22]
and chromatographic [23, 24] assays but the latter require a lot of sample processing, often
found to be destructive and not been able to provide much spatiotemporal profiling in native
biological system. As a result of continuous efforts, fluorescence based biomolecular
detection has grown as an efficient and attractive technique owing to its ease of handling,
more accuracy and better sensitivity. Recently, a number of excellent azide, nitro,
azamacrocyclic copper (II) complex and Michael acceptors based fluorescent probes have
been developed and used for detection of H
2
S [25-59]. However, the selective tracking and
real-time analysis of H S in environmental/biological samples is still a hurdle. This is because
2
of some of the reported probes are wrapped up with disadvantages such as lengthy multi-step
synthesis procedures with low yields, strong background fluorescence, low quantum yield
and notable affinity toward other biorelevant analytes such as cysteine, homocysteine,
glutathione etc. It has been well established that for being a smart molecular probe, small
3

molecular markers should not only display bright and fast fluorescence response but at the
same time should also exhibit long-range excitation/emission wavelengths (excitation
wavelength >400 nm and emission wavelength above 500 nm) to avoid awful issues
associated with cell damage and autofluorescence. Furthermore, large Stokes shift is another
crucial desirability that a smart molecular probe should own. It substantially
minimizes/diminishes the overlap between excitation and emission spectra leading to the
depletion of reabsorption of emitted photons which in consequence helps to get rid of
background interferences. Another advantage of having large Stokes shift is better sensitivity
and more accuracy in detection [27, 60-64]. Although several H S markers have been
2
witnessed in literature but the markers having large Stokes shift ( > 100 nm) are very small in
number and hence demands more attention [27, 30, 32-36, 39, 40, 46].
Carrying forward our research interests in developing smart molecular markers for H
2
S,[40,
4
1] herein, we wish to report our recent study on facile fabrication of an azide-based
fluorescent probe for the detection of H S chaperoned by 110 nm Stokes shift. 2-
2
azidoanthracene along with few of derivatives were synthesized and utilized for developing
various fluorophores or fluorescent chemical sensors, however, to our knowledge, application
of 2-azidoanthracene as a molecular marker for H
8]. It is worth mentioning that the developed probe exhibited excellent selectivity toward
S over various competitive sulfide derivatives.
2
S detection has not yet been reported [65-
6
H
2
2
. Experimental
2
.1.General information:
All chemicals were purchased from Merck, S.D. Fine and Sigma Aldrich, and were
used without any further purification. HPLC gradeDMSOwas used for the spectral
-
-
-
-, PO
3-
-
studies.Freshly prepared solutions of anions (Cl , F , Br , NO
3
4
and AcO as
-
-
-
2-
2-
+
+
tetrabutylammonium salt, I , HSO
3
, SCN , S
2
O
3
and S
2
O
5
as sodium salt), cations (Na , K ,
4

2
+
2+
Ca and Mg as nitrate salt), cysteine, homocysteine, glutathione and H
2
S (as NaHS) in H O
2
(1 mM HEPES, pH = 7.2) were used as stock solutions to record the UV-vis and fluorescence
1
spectra. FT-IR spectra were acquired in a Carry-660 FT-IR spectrometer in ATR mode. H
1
3
and C NMR spectra in DMSO-d were recorded on Jeol-ECX-500 MHz spectrometer using
6
tetramethylsilane as an internal standerd. Absorption spectra and fluorescence spectra were
recorded on PerkinElmer Lambda 750and Cary Eclipse spectrophotometer respectively.
HRMS-ESI spectra were recorded on a Bruker impact-HD spectrometer.
2.2.Synthesis:
Synthetic procedure for P4:P4 was synthesized following a previously known literature
report [65].2-Aminoanthracene (0.730 g, 3.77 mmol) was taken in water (10 mL) in a two-
necked round-bottom flask and stirred at room temperature for 5 min. The reaction mixture
was cooled to 0 °C and concentrated H
carefully with constant stirring. The whole reaction mixture was allowed to stir at 0 °C for 20
min followed by dropwise addition of NaNO (0.332 g, 4.82 mmol) in water (10 mL). The
resultant red coloured solution was stirred at 0 °C for 30 min. Next, NaN (439 mg, 6.76
2
SO
4
(5 mL) was added to the suspension very
2
3
mmol) was dissolved in water (10 mL) and added dropwise over 10 min to the previously
stirred mixture. The resulting solution mixture was then slowly warmed to room temperature
and stirred for another 3 h. Brown precipitation was observed, filtered through a sintered
funnel, washed with water (20 mL) and air dried for 1 h. The crude product was purified by
column chromatography (ethyl acetate/hexane = 2:8, v/v) to afford a yellow solid.
-
1
1
Yield: 59% (0.500 g). Melting point: 156-160 °C; FT-IR (ATR, ν in cm ): 2112-2130; H
NMR (DMSO-d6, 500 MHz, ppm): δ 8.58 (s, 1H), 8.52 (s, 1H), 8.15 (d, J = 8.9 Hz, 1H),
1
3
8
.07- 8.03 (m, 2H), 7.79 (s, 1H), 7.52 - 7.50 (m, 2H), 7.26 (dd, J = 8.9, 2.05 Hz, 1H); C
NMR (DMSO-d6, 125 MHz, ppm): δ 136.6, 131.9, 131.4, 130.9, 130.7, 129.1, 128.3, 127.9,
5

+
1
26.6, 126.3, 125.6, 125.1, 119.5, 115.1; HRMS: m/z calculated for C14
H
9
N [M] 219.0796,
3
+
found 219.0795;C14
H
9
N Na [M+Na] 242.0694, found 242.2885.
3
Synthesis procedure for P4′:Compound P4 (67 mg, 0.32 mmol) was dissolved in THF
followed by addition of NaHS solution (119 mg, 2.14 mmol) in 3 mL H O dropwise with
2
constant stirring at room temperature. The reaction mixture was further stirred at room
temperature for 2 h and the solvent was removed under reduced pressure. The obtained
residue was purified by column chromatography (ethyl acetate /hexane = 4:96, v/v)to
furnishP4' as yellow solid.
-
1
1
Yield: 61% (35 mg); FT-IR (ATR, ν in cm ): 3444, 3361 ; H NMR (DMSO-d
6
, 500 MHz,
ppm): δ 8.27 (s, 1H), 8.02 (s, 1H), 7.90 - 7.85 (m, 2H), 7.80 (d, J = 8.9 Hz, 1H), 7.37 - 7.34
13
(
m, 1H), 7.29 -7.26 (m, 1H), 7.04 (dd, J = 8.95, 2.05 Hz, 1H), 6.88 (s, 1H), 5.57 (s, 2H); C
NMR (DMSO-d6, 125 MHz, ppm): δ 146.09, 133.69, 131.94, 128.99, 128.43, 128.13, 127.07,
1
26.57, 125.83, 125.17, 123.12, 120.96, 120.90, 102.72; HRMS: m/z calculated for C14
12
H N
+
[
M+H] 194.0970, found 194.0964.
2
.3.UV-vis and fluorescence titrations:
UV-vis and fluorescence titrations were conducted using 50 µM solutions of probe (P4) in
DMSO: H O solution (1:1, v/v).H O was used in form of 1 mM HEPES buffer of pH = 7.2.
2
2
All measurements were performed using 395 nm as an excitation wavelength and keeping
both excitation and emission slit widths as 5at 37 °C. UV-vis and fluorescence spectra were
recorded using 3 mL quartz cuvette (path length 1 cm) by addition of aliquots of freshly
-
-
-
-
-, PO
3-
, AcO , HSO
-,
-
prepared stock solutions of various analytes such as Cl , F , Br , I , NO
3
4
3
-
2-, S
2-
+
+
2+
2+
SCN , S
2
O
3
2
O
5
, Na , K , Ca , Mg , cysteine, homocysteine and glutathione. NaHS
solution was used as a source of H
2
S (50 mM stock solution).UV-vis and fluorescence
6

titrations were conducted taking aliquots of highly concentrated stock solutions of various
analytes to avoid dilution error.
2
.4.DFT calculations
The geometry of the compounds (P4 and amine P4′) was optimized at density functional
theory (DFT) with B3LYP/6-31G(d) basis set [69, 70] with no symmetry constraint using
Gaussian 09 suite of programmes [71]. Frequency calculation at the same level with the same
basis set was performed to ensure that the geometries correspond to real minima. The solvent
effect was incorporated employing conducting polarisable continuum model (CPCM model)
[
72]. Water was chosen as solvent to optimize the geometry and for TD-DFT calculation.
Gauss view software along with Chemcraft was used for data visualization and preparation
purpose [73].
3
. Results and discussion
3
.1. Synthesis
Probe P4 was synthesized in a single step by using 2-aminoanthracene following the
experimental protocols as outlined in Scheme 1. The chemical structures of P4 and P4′were
characterized using various spectroscopic techniques such as FT-IR, NMR spectroscopy and
high-resolution mass spectrometry (HRMS).
3.2.Photo-physical studies
The photophysical properties ofP4 were investigated by absorption/emission
spectroscopy in 50% aqueous DMSO solution buffered with HEPES (1 mM, pH = 7.2). As
-5
shown in Fig. 1, initially P4(5 x 10 M) showed one strong absorption band centred at
2
76nmand a broad band ranging from 314 nm to 431 nm having peaks at 341, 357, 373,
3
94and 418 nm with variable intensity due to π-π* and n-π* transitions of aromatic rings and
-5
the azide (-N
3
) group respectively. Upon introduction of H S (50 x 10 M), 13 nm blue shift
2
in 276 nm was observed along with even broader absorption band ranging from 315 to 470
7

nm (absorption peaks at 321 nm, 338 nm, 355 nm, 374 nm, 394 nm and 418 nm) (Fig. 1, S1).
On the contrary, the absorption profile of P4 remained almost unaltered in the presence of
-
-
-
-
-, PO
Na , K , Ca , Mg , Cys, Glu and Homo Cys (Fig. 1a).
In the absence of H S, P4 was almost non-fluorescent in nature (Fig. 2). The silent optical
3-
-
-
-
2-, S
O
2-,
various bio-analytes such as Cl , F , Br , I , NO
3
4
, AcO , HSO
3
, SCN , S
2
O
3
2
5
+
+
2+
2+
2
behaviour was attributed to the photoinduced electron transfer (PET) from excited
fluorophore (anthracene) to the strong electron-withdrawing group (azide unit) which is also
popularly known as donor-excited- PET (d-PET)[28].Upon exposure to H S,a significant
2
fluorescence turn-on response was observed at 505 nm (quantum yield: 0.274, quinine
sulphate was used as standard)due to the stoppage of d-PET process. The fluorescence
response was guided by 110 nm Stokes shift (Fig. 3).
H
2
S instigated fluorescence enhancement and occurrence of multiple isobestic points (268,
91, 341, 384 and 398 nm) (inset Fig. 1b) directed us to envision the reduction of azide (P4)
to amine (2-aminoanthracene, P4′) in the presence of H S.It is important to note that P4 was
allowed to react with H S at 37 °C for 40 minutes to achieve optimum response (Fig. S2).
This is due to the slower rate of H S induced reduction at room temperature (25 °C) (Fig. S2).
2
2
2
2
Further, time dependent fluorescence response experiment was conducted to monitor the
-5
reduction kinetics of azide group of P4in the presence of NaHS (50 x 10 M). Although
enhancement in fluorescence intensity was instantaneous to the addition of NaHS (~10%
enhancement), it took around 40 min to obtain a stable/saturated fluorescence signaling (Fig.
2
b). The pseudo first order rate constant (kobs) and time required for 50% completion (t1/2) of
-3
−1
the reaction was found to be 1.26 x 10 sec and 550 sec respectively (Fig. S3). This data
revealed the quick response time of P4 toward H S along with total time required for
2
complete reduction of azide group.
8

Fascinated by the initial photophysical outcomes, we became curious to check whether any
other relevant analyte has the similar potential of displaying the fluorescence enhancement
-
-
-
brought by H S. In this regard, probe P4 was treated with wide variety of anions (Cl , F , Br ,
2
I-, NO
biothiols (homocysteine, glutathione and cysteine).Satisfyingly, probe P4 remained silent in
the presence of all those analytes, however it displayed bright green emission once H S was
added to the solution (Fig. 4, S4). Therefore, probe P4 established its high selectivity toward
S over other various relevant competitors. The mechanistic pathway for H S instigated
reduction of 2-azidoanthracene (P4) was adopted from a recent report by Pluth Group
-, PO
3-
, AcO , HSO
-
-
, SCN , S
-
O
3
2-
and S
O ), cations (Na , K , Ca and Mg ) and
2-
+
+
2+
2+
3
4
3
2
2
5
2
H
2
2
(Scheme 2) [56]. Reduction of azide functionality to an amine one is chiefly controlled by
-
high nucleophilicity of HS and its subsequent attack at the electrophilic group like -N
3
[56].
-
Besides being the smallest unit of thiol community, HS could also approach the electrophilic
centre overlooking any kind of steric hindrance unlike other thiols such as cysteine,
homocysteine and glutathione [59]. Next, limit of detection (LOD) and limit of quantification
values (LOQ) were calculated using 3σ/slope and 10σ/slope methods [74] and those values
were found to be 1.19 µM and 3.99 µM respectively (Fig. S5), which strongly validated the
sensitivity of P4 toward H S.
2
3
.3.Spectroscopic investigations for azide reduction
Further to corroborate our assumption that exposure of H
2
S led to the reduction of P4 to P4′,
1
1
H-NMR and mass spectroscopic studies were carried out. During H-NMR experiments,
appearance of a sharp resonance signal at 5.56 ppm in the isolated product after exposure of
H
2
Swas attributed to the –NH protons of P4′(Fig. 5).It was further supported by the similar
2
NMR spectra of anthracene amine(Fig. S6, S7).
9

Similarly, the mass spectra of P4 in the presence of H S showed the disappearance of
2
molecular ion peak at 219.0795, sodium adduct peak at 242.2855 and the appearance of
+
distinct peak corresponding to anthracene amine at 194.0964 [M+H] (Fig. S8). In
1
combination, the outcomes of H-NMR and mass experiments strongly supported our
speculation that the azide functionality of P4 underwent reduction to produce the amine
derivative P4′ (Scheme 2).
3
.4.Theoretical studies
Finally, to understand the probable structures of P4, P4′ and simulate their absorption spectra
in water as a polar medium, theoretical analyses were carried out employing density
functional theory (DFT). Optimized geometry of P4 and the frontier molecular orbitals are
depicted in Fig. 6a. While the HOMO-1, HOMO and LUMO are distributed throughout the
P4 derivative, LUMO+1 is located on the azido functionality (Fig. 6a). This is due to the
electron withdrawing effect of azido functionality which facilitates electron transfer from the
anthracene moiety to the azido functionality resulting in generation of non-fluorescent azido
derivative. In the continuation, geometry of the amine derivative was also optimized (Fig. 6b)
and the contribution of molecular orbitals was evaluated. HOMOs and LUMOs are
distributed throughout the amine containing anthracene system (Fig. 6b). Time dependent-
DFT calculations (TD-DFT) have been performed to correlate the absorption maxima of the
azide and amino derivative (Fig. S9, S10 and Table S1). Theoretically simulated absorption
profiles of P4/P4′ were in close agreement with the experimentally observed absorption
spectra (Fig. S9, S10 and Table S1).
4
. Conclusions
10

To conclude, we have successfully developed following a simple one step synthesis process
an anthracene-based molecular probe (P4) for the detection of H S. LOD and LOQ values for
S sensing were found to be 1.19 µM and 3.99 µM respectively. The probe P4 was found to
be non-fluorescent initially due to d-PET process. Interaction with H S led to the reduction of
2
H
2
2
azide functionality to the amine which in turn blocked the fluorescence quenching through d-
PET process and generated the concealed emission. This emerging bright green emission was
also chaperoned by 110 nm Stokes shifts. The H S instigated azide reduction was evidenced
2
by different spectroscopic studies and photo-physical outcomes were further validated by
TD-DFT calculations. P4 being a non-emissive molecular material, background fluorescence
issue was successfully eliminated. We strongly feel that the probe P4 could be very handy,
highly selective and efficient for monitoring H
biological samples.
2
S level in various environmental and
Acknowledgement
Financial support was received from the Department of Science and Technology, India
(
Grant No. SERB/F/2408/2012-13). SS is thankful to the National Project Implementation
Unit, MHRD for TEQIP-III faculty programme. We thankfully acknowledge the Director, IIT
Mandi for research facilities. The support of Advanced Materials Research Center (AMRC),
IIT Mandi, for sophisticated instrument facility is thankfully acknowledged. We are thankful
to the reviewers for their valuable suggestions.
11

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[
20

-
3
2
2
1
1
0
0
.0
.5
.0
.5
.0
.5
(a)
P4 + Br
P4, P4 + Other analytes
0
.25
-
P4 +Br
0.20
P4 + NaHS
0.15
P4, P4 +Other analytes
P4 +NaHS
0.10
0.05
0.00
300
350
400
450
500
Wavelength(nm)
.0
2
50
300
350
400
450
500
Wavelength (nm)
2
2
1
1
0
0
.5
.0
.5
.0
.5
.0
P4
(b)
0.20
P4 + NaHS
0.15
0.10
0.05
0.00
350
400
450
500
Wavelength (nm)
2
50
300
350
400
450
500
Wavelength (nm)
-
5
Fig. 1. (a) UV-vis spectra of probe P4 (5 x 10 M) in DMSO : H
2
O (1:1, v/v) upon addition
-
-
-
-
-
3-
-
-
-
2-
2-
+
+
2+
of NaHS, Cl , F , Br , I , NO
3
, PO
4
, AcO , HSO
3
, SCN , S
2
O
3
, S
2
O
5
, Na , K , Ca ,
2
+
-5
-5
Mg ,Cys, Glu and Homo Cys (50 x 10 M); (b) UV-vis spectra of probe P4 (5 x 10 M) in
DMSO : H O (1:1, v/v) upon addition of NaHS (50 x 10 M).
-5
2
21

700
600
500
400
300
200
100
0
NaHS (0-10 equiv)
(
a)
450
500
550
600
650
700
Wavelength (nm)
7
6
5
4
3
2
1
00
00
00
00
00
00
00
0
0
to 40 min
(b)
6
5
4
00
00
00
300
2
1
00
00
0
0
10
20
30
40
Time (Min)
4
50
500
550
600
650
700
Wavelength (nm)
-
5
Fig. 2. (a)Fluorescence responses of probe P4 (5 x 10 M) in DMSO : H
2
O (1:1, v/v) upon
-
5
addition of increasing quantities of NaHS (0 to 50 x 10 M). The solution was incubated for
22

4
0 min at 37 °C (λex=395 nm, λem= 505 nm); (b) Fluorescence responses of probe P4 (5 x 10-5
-5
M) to NaHS (50 x 10 M) after incubation of 0 to 40 min in DMSO : H O (1:1, v/v).
2
23

Absorbance
Emission
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
(a)
Excitation
Emmision
(b)
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
2
50 300 350 400 450 500 550 600 650 700
3
90
455
520
585
650
Wavelength (nm)
Wavelength (nm)
-
5
Fig. 3. (a) Normalized absorption (black) and emission spectra (blue) of probe P4 (5 x 10
-5
M) in DMSO: H
blue) and emission spectra (green) upon addition of NaHS (50 x 10 M). The solution was
incubated for 40 min at 37 °C(λex=395 nm, λem= 505 nm).
2
O (1:1, v/v) upon addition of NaHS (50 x 10 M); (b) normalized excitation
-5
(
24

7
6
5
4
3
2
1
00
00
00
00
00
00
00
0
P4 + NaHS
P4, P4 + Other analytes
4
00
500
600
700
Wavelength (nm)
-
5
Fig. 4. Fluorescence responses of probe P4 (5 x 10 M) in DMSO : H
2
O (1:1, v/v) upon the
-
-
-
-
-
3-
-
-
-
2-
2-
+
+
addition of NaHS, Cl , F , Br , I , NO
3
, PO
4
, AcO , HSO
3
, SCN , S
2
O
3
, S
2
5
O , Na , K ,
2
+
2+
-5
Ca , Mg ,Cys, Glu and Homo Cys (50 x 10 M). Inset: Fluorescence intensity of P4 in the
presence of different analytes. A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q and R
-
-
-
-
-
3-
-
2-
, SCN , HSO
-,
-
representsCys, Glu, Homo Cys, F , Cl , Br , I , NO
3
, PO
, Na , K , Ca and Mg . The solution was incubated for 40 min at 37 °C (λex = 395
nm, λem = 505 nm).
4
, AcO , S
2
O
3
3
2
-
+
+
2+
2+
S
2
O
5
25

1
Fig.5. H-NMR spectra of 2-aminoanthracene, P4 and P4′ (isolated product of P4 with
NaHS) in DMSO-d
6
.
26

HOMO-1
HOMO
HOMO-1
-5.99 eV
HOMO
-5.02 eV
-6.38 eV
-5.36 eV
LUMO
1.89 eV
LUMO+1
-1.12 eV
LUMO
-1.61 eV
LUMO+1
-0.24 eV
-
(a)
(b)
Fig.6. Optimized structures and frontier molecular orbitals of P4 (a) andP4′ (b) (isocontour at
.03 au).
0
27

Scheme 1. Synthesis of P4 and P4'
28

Scheme 2.Proposed mechanism of azidereduction by NaHS
29