Cell-Permeable Fluorogenic Probes for Identification and Imaging Nitroreductases in Live Bacterial Cells

Article abstract of DOI:10.1021/acs.joc.8b02746

Enzyme-activated fluorogenic probes, which invoke enzymatic catalysis to trigger the generation of fluorescence, provide a versatile platform for monitoring biological processes. The development of fluorogenic probes that can readily penetrate the cell envelopes of bacteria are essential to examine intracellular targets of live bacterial cells. Herein, we present the design, synthesis, properties, and biological applications of two series of fluorogenic probes based on cyanine 5 for identification of bacterial nitroreductase (NTR). The selected fluorogenic probe 3 generates a rapid 10-fold fluorescence response after being catalytically reduced by NTR to the intermediate para-aminobenzyl substituted which then underwent a rearrangement elimination reaction. Moreover, probe 3 is cell permeable for both Gram-positive and Gram-negative bacterial cell envelopes and is selective for NTR over other biological analytes, thus minimizing the background signal and enabling the real-time intracellular imaging of NTR in live bacterial cells.

Full text of DOI:10.1021/acs.joc.8b02746

Subscriber access provided by UNSW Library
Article
Cell-permeable Fluorogenic Probes for Identification
and Imaging Nitroreductases in Live Bacterial Cells
Yubin Ji, Yali Wang, Na Zhang, Shengnan Xu, Leilei
Zhang, Qinghua Wang, Qingyang Zhang, and Hai-Yu Hu
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02746 • Publication Date (Web): 27 Dec 2018
Downloaded from http://pubs.acs.org on December 31, 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 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
Cell-permeable Fluorogenic Probes for Identification and Imaging
Nitroreductases in Live Bacterial Cells
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Yubin Ji†, ‖, Yali Wang†, ‡, ‖, Na Zhang†, ‡, ‖, Shengnan Xu‡, Leilei Zhang‡, Qinghua Wang‡, Qingyang Zhang‡,
* and Hai-Yu Hu‡, *
†School of Life and Environment Sciences, Harbin University of Commerce, Harbin 150076, China
‡State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia
Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
Corresponding Authors* E-mail: haiyu.hu@imm.ac.cn; E-mail: zhangqingyang@imm.ac.cn
‖Y. J., Y. W. and N. Z. contributed equally to this work.
Abstract Graphic
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 34
1
2
3
4
5
Abstract
6
7
8
9
Enzyme-activated fluorogenic probes, which invoke enzymatic catalysis to trigger the generation of
fluorescence, provide a versatile platform for monitoring biological processes. The development of
fluorogenic probes that can readily penetrate the cell envelopes of bacteria are essential to examine
intracellular targets of live bacterial cells. Herein, we present the design, synthesis, properties, and biological
applications of two series of fluorogenic probes based on cyanine 5 for identification of bacterial
nitroreductase (NTR). The selected fluorogenic probe 3 generates a rapid 10-fold fluorescence response after
catalytically reduced by NTR to the intermediate para-aminobenzyl substituted which then underwent a
rearrangement elimination reaction. Moreover, probe 3 is cell-permeable for both Gram-positive and
Gram-negative bacterial cell envelopes, and is selective for NTR over other biological analytes, thus
minimizing the background signal and enabling the real-time intracellular imaging of NTR in live bacterial
cells.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Introduction
Fluorescence microscopy is an essential tool for understanding and exploring the mechanisms governing
cellular processes at the molecular level1. Despite the rapid development of techniques to study bacteria, the
largely impenetrable cell envelope of these organisms present major challenges in imaging experiments2.
The ideal probe for bacterial imaging would encompass many favorable properties such as fast response,
high brightness, far-red emission and excitation wavelengths, enhanced sensitivity and selectivity for targets,
and suitability for biological applications3. The cyanine 5 (Cy 5) fluorophore is a useful fluorescent
molecule, being applicable to biological investigations utilizing fluorescence microscopy, owing to its
excellent photophysical properties in aqueous media, including outstanding molar extinction coefficient,
aqueous solubility and tolerance to physiological pH (pH 3-10), long excitation and emission wavelengths
ACS Paragon Plus Environment

Page 3 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
(620-670 nm), excellent photostability and high quantum yield, and its wide use as a fluorescent tag4.
However, it has not been widely employed as bacterial fluorogenic probes, which offer the feature of
fluorescence turn-on response to the biological analytes without the need to remove or wash off the
unreacted probes5. One of the reasons for this is probably the lack of a good strategy to improve the bacterial
cell envelope permeability and regulate the fluorescence of the Cy 5 fluorophore6. Therefore, a novel
strategy to overcome bacterial cell envelopes penetration barrier meanwhile regulate the fluorescence of the
Cy 5 fluorophore would potentially be applicable to the design of fluorogenic probes for a wide range of
intracellular bacterial analytes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A number of strategies have been reported to develop fluorogenic probes based on cyanine fluorophore,
such as fluorescence resonance energy transfer (FRET) mechanism7, the modulation of the pi-conjugation8
and photoinduced electron transfer9. Another common strategy is to chemically cage the fluorophore with a
group that can be specially removed by the analyte10. However, so far these caging strategies are hampered
by limited functionalization position of the cyanine chromophore, either at the polyenic methine chain or at
the alkyl side chains, and limited caging group variability. In this work, we report two novel series of
fluorogenic probes based on Cy 5 with various lipophilic nitroaromatic moieties as caging groups. Instead of
functionalizing at the alkyl side chains, the caging groups are decorated at the benzene unit and the methine
backbone of Cy 5 core. In addition, the introduction of lipophilic nitroaromatic moieties make the
cell-impermeable Cy 5 could penetrate both Gram-positive and Gram-negative bacterial cell envelopes. The
selected fluorogenic probe 3 was successfully applied for selective identification and imaging NTR in live
bacterial cells (Figure 1).
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 34
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. NTR-triggered process of probe 3 in live bacterial cells.
Results and Discussion
Nitroreductases (NTRs), are a family of flavin-containing enzymes, which metabolize nitrosubstituted
compounds using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). NTRs are reported
biomarker for tumor and bacterial infection detections11. Previously, we reported an NTR-triggered
fluorescent probe by conjugating nitro-imidazole group to the side chain of Cy 5.5, which exhibited fast and
sensitive response to NTR, though it is unclear whether this approach can be generalized for other cyanine
fluorophores12. Herein, we studied a novel caging and uncaging strategy by attaching lipophilic
nitroaromatic substitutions to Cy 5 fluorophore core structure and designed probes specific for NTR. To
improve the fluorescence response to NTR and increase the membrane permeability of probe, we
synthesized two series of eight novel probes by introducing different lipophilic nitro groups to R1 or R2
positions of Cy 5 carboxyl acid core, respectively (Figure 2). By incorporating nitro group, nitrobenzene
group, nitroaromatic carbamate moieties into the benzene unit of Cy 5 respectively, the obtained probes 1-4
have an insight into the influence of group lipophilicity and arrangement. Similarly, we obtained probes 5-8
by introducing nitrobenzene group, nitroaromatic carbamate moieties and nitroaromatic ester fragment to the
methine backbone of Cy 5, and explored the influence of connecting position of Cy 5 core.
ACS Paragon Plus Environment

Page 5 of 34
The Journal of Organic Chemistry
1
2
3
4
R1
5
6
7
N
N
core
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
N
R3
N
probe 1-4
OH
Cell-permeable
side chain
O
N
N
OH
R2
Cy 5 carboxyl acid
O
probe 5-8
OH
Probe 1-4: R1 = lipophilic nitroaromatic moieties, R3 = Methyl
Probe 5-8: R2 = lipophilic nitroaromatic moieties, R3 = Ethyl
Figure 2. Design strategies of NTR-triggered membrane permeable probes based on cyanine 5.
The heterocyclic ammonium salts (10a, 10b, 10c and 10d) were synthesized by quaternization of the
corresponding heteroaromatic base 2,3,3-trimethylindolenine (9a, 9b and 9c) with suitable alkylating agents,
including 6-bromohexanoic acid and alkyl halides in o-dichlorobenzen or acetonitrile. The three-carbon
spacer precursors 11 and 12 were prepared from the commercially available reagents tetramethoxypropane
and dibromo-4-oxobut-2-enoic acid, respectively (Scheme 1). Probe 1-8 were synthesized as depicted in
Scheme 2. Three-carbon spacer precursor 11 as starting material on reacting with precursor 10a gave an
intermediate without further purification, which on condensation with precursors 10b, 10c furnished
asymmetrical cyanine probe 1 and intermediate 13, respectively. Finally, cyanine intermediate 13 on reacting
with different nitroaromatic boric acid reagent (Scheme S1) via Suzuki-Miyaura conditions resulted in the
formation of probe 2-4 in good yields. In a similar way, cyanine intermediate 14 was prepared from the
precursor 12 in good yield. The final probe 5-8 were obtained via Suzuki-Miyaura conditions with
corresponding nitroaromatic boric acid reagent (Scheme S1). All the obtained products were characterized
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 34
1
2
3
4
5
6
7
8
9
1
by H NMR, 13C NMR and HRMS (Supporting information). We expected the selective catalytic reduction
of the probes by NTR would result in an off-on fluorescence response.
Scheme 1. Synthesis of precursorsa
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R
R
i
N
N
R3
9a
9b
9c
: R = H
: R = NO2
: R = I
10a
10b
10c
10d
: R = H, R3 = 5-carboxypentyl
: R = NO2, R3 = methyl
: R = I, R3 = methyl
: R = H, R3 = ethyl
Cl
H
N
H
N
O
O
O
ii
O
11
Br
Br
O
H
N
H
N
Br
iii
HO
H
O
Br
12
aReagents and conditions: (i) 6-bromo hexanic acid, bromine ethane, iodomethane, o-dichlorobenzen or
acetonitrile, 120 oC for 24 h; (ii) aniline, HCl, H2O, 50 oC for 30 min; (iii) aniline, ethanol, 60 oC for 4 h.
Scheme 2. Synthesis of probe 1 (a), 2-4 (b), 5-8 (c)a
O2N
a
N
Br
O2
N
OH
N
10a
N
I
Cl
N
H
O
I
10b
N
N
H
i
ii
11
1
probe
O
OH
R1
I
N
b
Br
I
OH
N
I
10a
N
N
O
I
Cl
I
10c
N
N
N
N
H
H
i
iv
iii
2-4
probe
11
13
O
O
OH
OH
O
NO2
NO2
O
O
NO2
4
R1
probe
:
=
2
R1
3
R1
O
probe
:
=
probe
:
=
NH
NH
c
N
Br
H
H
N
Br
Br
N
N
Br
N
N
10d
Br
Br
OH
10a
vi
N
N
R2
Br
O
v
vii
12
5-8
14
probe
O
O
OH
O
OH
O
NO2
NO2
O
7
R
=
2
probe
:
O
5
R
NO2
probe 6: R2
=
NH
probe
:
=
8
R
probe
:
=
2
2
O
O
NO2
NH
ACS Paragon Plus Environment

Page 7 of 34
The Journal of Organic Chemistry
1
2
3
4
o
aReagents and conditions: (i) compound 11 and compound 10a, AcOH/(Ac)2O, 130 C, 2 h, evaporate; (ii)
5
6
7
then with compound 10b, pyridine/AcOH, 120 oC, 2 h; (iii) with compound 10c, pyridine/AcOH, 120 oC, 2
8
9
o
h; (iv) different nitroaromatic boric acid reagents, Pd(dppf)2Cl2, K2CO3, 1,4-dioxane, H2O, 90 C, 1.5 h; (v)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
compound 12 and compound 10d, (Ac)2O, 60 oC, 4 h; (vi) then with compound 10a, NaOAc, 50 oC, 2 h; (vii)
different nitroaromatic boric acid reagents, Pd(dppf)2Cl2, K2CO3, 1,4-dioxane, H2O, 90 oC, 1.5 h.
With these compounds in hand, the fluorescence responses of these probes (probe 1-8) to purified E. coli
NTR were first investigated in 0.05 M Tris buffer solution containing 1.5% DMSO (pH 7.4). As depicted in
Figure 3 and S1, only probe 1 and 5 showed intense absorption peak with their maximal absorbance at
around 645 nm, whereas the other probes exhibited low smooth absorption peak caused by molecular
aggregation and poor solubility in above solution. These results indicated that increasing lipophilicity of the
probes leads to molecular aggregation and decreased solubility of the probes in aqueous environment.
However, a drastic enhancement of the absorption intensity of these probes was observed after incubation
with NTR in the presence of NADH. In the fluorescence emission spectra, the fluorescence intensity of the
probes was very low except for probe 5 due to its nice aqueous solubility. Moreover, on adding 5 μg/mL
NTR in the presence of NADH (500 μM), no significant changes were observed for probe 1, 2, 4, 5 and 6,
while obvious fluorescence enhancement were observed for probe 3, 7 and 8 (about 10-fold, 5-fold and
5-fold, respectively). To clarify the reasons for the fluorescence activation differences of probe 1-8 to NTR,
we first investigated the reduction response of probe 1-8 to NTR in vitro by HRMS after 1-hour incubation
with the enzyme. As shown in Figure S3, probes 2, 4 and 5 could be hardly or not reduced, while the probe
1, 3, 6, 7 and 8 could be reduced and their corresponding amino reduced products were detected after NTR
incubation. To clarify the reason for the fluorescence inactivation of probe 1 to NTR, the absorption and
fluorescence spectra of chemical synthesized the reduction product of probe 1, product 18, were
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 34
1
2
3
4
5
6
7
8
9
investigated. As shown in Figure S1, product 18 showed low smooth absorption and fluorescence emission
in Tris buffer which led to no fluorescence enhancement after probe 1 reduced by NTR. The absorption and
fluorescence emission properties of product 19, NTR reduction product of probe 2, and product 21, reduction
product of probe 5 and probe 6, in Tris buffer were also studied. Product 19 exhibited good absorption and
fluorescence emission properties, however, only very small amount of product 19 could be detected in the
HPLC after 1-hour incubation of probe 2 with NTR (Figure S4), which resulted in only a little fluorescence
recovery of probe 2. UV-vis and fluorescence emission intensity of product 21 revealed it was not
fluorescent in Tris buffer (Figure S1i and k), which might because of the a-PET process. Moreover, LC-MS
analysis showed strong retention peak of NTR reduction product of probe 3 after 1-hour treatment with NTR
(Figure S3e, f and S4b), and chemical synthesized reduction product of probe 3, product 20, shows strong
absorption and fluorescence emission intensity (Figure S1c). These results indicated that probe 3 has the
best NTR detection potentialities.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In order to clarify the fluorescence quenching of probe 3 is induced by photoinduced electron transfer
(PET) progress or its aggregations in Tris buffer, we calculated the free-energy changes (ΔGeT) by cyclic
voltammetry studies and measured the UV-Vis absorption and fluorescence emission spectra of probes 3 in
organic solvent in DMSO and Tris buffer, respectively, and compared with the spectra of product 20. As
shown in Figure S5, the nitroaromatic moiety (25) act as an electron acceptor for Cyanine fluorophore (24)
in probe 3. According Rehm-Weller equation, we got ΔGeT < 0 of probe 3 by cyclic voltammetry, implying
d-PET process could happen (Table S1). Next, probe 3 displayed stronger absorption and fluorescence
intensities in DMSO than in Tris buffer, which reveals that probe 3 has a typical aggregation-caused
quenching (ACQ) effect in Tris buffer. Moreover, Probe 3 and product 20 exhibited similar absorbance
spectra in DMSO; however, the fluorescence intensity of probe 3 showed 15% lower than product 20.
ACS Paragon Plus Environment

Page 9 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Therefore, we inferred that ACQ effect and d-PET process quenches probe 3 emission fluorescence
synergistically, and the ACQ effect is the major cause for fluorescence quench of probe 3. Furthermore, the
primary amine of product 20 is considered to be an electron rich group which may quench fluorescence by
a-PET process and protonation could prevents this electron transfer. To verify whether the product 20 has an
a-PET process or not, ΔGeT of product 20 and UV-vis and fluorescence emission spectra of product 20 in
DMSO and different pH buffer were estimated. The result shows ΔGeT > 0, indicating the PET process from
primary amine to the excited Cy chromophore was unlikely happened. As shown in Figure S6, product 20
showed intense absorption peak and emission intensity in DMSO. This result implied unprotonated product
20 itself has excellent fluorescence properties. Moreover, compared with probe 3, product 20 displayed
significant absorption and fluorescence emission in Tris buffer which indicated that product 20 has
improving solubility in Tris buffer. We evaluated the pH dependence of the fluorescence emission intensity
of product 20 in PBS (pH 6.0-8.0) and Tris buffer (pH 7.0-9.0), respectively. Product 20 exhibited
significant and similar fluorescence in PBS with different pH values (pH 6.0-8.0) (Figure S7). And in tris
buffer (pH 7.0-9.0), the fluorescence intensity decreases slightly with the increase of pH values (Figure 4a).
These results indicated that protonation of amino group did not significantly enhance the fluorescence
intensity and the primary amino group could not transfer an electron to the excited cyanine chromophore and
quench its fluorescence.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. (a) Absorption and (b) fluorescence emission spectra of 5 µM probe 3 (λex = 620 nm). (i) (Green line):
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 34
1
2
3
4
5
6
7
8
9
probe 3 in pH 7.4 Tris buffer (control); (ii) (blue line): the system (i) + 500 μM NADH (another control); (iii) (red
line): the system (ii) + NTR (5 μg/mL); (iv) (purple line): product 20 in pH 7.4 Tris buffer. Spectra were measured
after reaction of the probe with NTR for 1 h at 37 °C in 0.05 M Tris buffer solution containing 1.5% DMSO (pH
7.4).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Furthermore, the reduction conditions in terms of pH value and temperature of probe 3 by NTR also
revealed that the probe can work effectively under the physiological condition (pH 7.4, 37 °C) (Figure 4a
and 4b). Subsequently under the optimized conditions, the fluorescence responses of probe 3 to varied
concentrations (1.0-5.0 µg/mL) of NTR were investigated. As depicted in Figure 4c, a gradual increase in
fluorescence intensity was observed with increase in the NTR concentrations, and good linearity between the
fluorescence intensity and the NTR concentrations in the range of 1.0-5.0 µg/mL was observed, with an
equation of F = 344.7 C (µg/mL) + 180.0 (R = 0.9903) (Figure 4d). The detection limit of the probe for
NTR was determined to be 32.9 ng/mL.
Figure 4. Effects of (a) pH and (b) temperature on the fluorescence of probe 3 (5 µM) reacting with NTR.
ACS Paragon Plus Environment

Page 11 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Red line: probe 3 (5 µM) + NTR (5 µg/mL) + NADH (500 µM) in 0.05 M Tris buffer for 1 h at 37 °C; black
line: probe 3 (5 µM) + NADH (500 µM) in 0.05 M Tris buffer for 1 h at 37 °C; blue line: product 20 (5 µM)
in 0.05 M Tris buffer. (c) Fluorescence emission spectra of probe 3 (5 μM) reacting with different
concentrations of NTR (1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 μg/mL) in the presence of 500 μM NADH at 37
oC for 1 h. (d) Linear correlation between the concentration of NTR and the fluorescence intensity of the
reaction mixture, data shown as mean ± SD (n = 3).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In order to further study the specificity of probe 3 to NTR, we examined various potential biorelevant
interferents, such as reductive endogenous thiols (Cys, DTT, GSH and homocysteine), arginine (Arg),
ascorbic acid (Vc), glucose, reactive oxygen species (H2O2, ClO-), and inorganic salts (MgCl2, CaCl2, KCl
and NaCl). As can be seen from Figure 5a, when these potential interfering species were added to probe 3 in
the presence of 500 μM NADH, no obvious fluorescence enhancement was observed, including reductive
endogenous thiols at a high concentration. Remarkably, when NTR (5 µg/mL) was added, the significant
fluorescence enhancement was observed as before. These results demonstrated that probe 3 shows excellent
specificity for NTR over other species and hence can be used for NTR detection.
(a)
2000
1500
1000
500
0
(b)
2500
2000
1500
1000
500
c
d
e
a,b
0
-
+
+
2
a
+
+
a
2
s
c
e
3
y
g
r
O 2
660
680
700
720
740
T
R
H
s
o
c
y
e
c
O
V
K
S
l
g
T
T
b
C
N
A
H
H 2
C
o
C
D
N
G
M
u
r
l
p
g
Wavelength / nm
Figure 5. (a) Fluorescence responses of probe 3 (5 µM) to various species: NTR (5 µg/mL), Cys (1 mM), DTT (1
mM), GSH (1 mM), Hcy (1 mM), Arg (1 mM), Vc (1 mM), glucose (10 mM), ClO- (10 µM), H2O2 (10 µM),
MgCl2 (2.5 mM), CaCl2 (2.5 mM), KCl (10 mM) and NaCl (10 mM). (b) Fluorescence emission spectra of probe 3
(5 µM) in the different reaction systems. a: probe 3 in pH 7.4 Tris buffer (control); b: the system a + 500 μM
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 34
1
2
3
4
5
6
7
8
9
NADH (another control); c: the system b + NTR (5 μg/mL); d: the system c + dicoumarin (2.5 μM); e: the system
c + dicoumarin (5.0 μM). All spectra were acquired in 0.05 M Tris buffer solution containing 1.5% DMSO (pH
7.4) for 1 h at 37 °C. λex/λem = 620/657 nm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Furthermore, an enzyme inhibition experiment also proved the fluorescence enhancement was caused by NTR.
As shown in Figure 5b, when probe 3 was pre-treated with dicoumarin, a common inhibitor of NTR14, and then
mixed with NTR, the fluorescence intensity was much lower than that without dicoumarin. Moreover, with the
increase of dicoumarin concentration, the fluorescence intensity of the mixture of probe 3 and NTR decreased
progressively. These results confirmed that the NTR activity was effectively inhibited by dicoumarin and thus the
fluorescence off-on response of probe 3 to NTR indeed derives from the enzyme-catalyzed reduction reaction.
Moreover, before using probe 3 for its potential applications in bioimaging, time-dependent fluorescence intensity
test of probe 3 in vitro was estimated to further understand cellular uptake and substrate reduction. E. coli cells
were firstly treated with 10 μM of probe 3 at 37 °C. After incubation for 1 h, the cells were washed three times
with Tris buffer, and then continue incubating in Tris buffer for 0-24 h. As shown in Figure S8, prolonging the
incubation time led to a progressive increase in fluorescence intensity, confirming that substrate reduction is the
key step in the appearance of fluorescence. Further cytotoxic properties were also tested against Hela cells by the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays. As
seen from Figure S9, there was no obvious cytotoxicity when cells were incubated with probe 3 for 24 h at a
concentration of 50 μM.
Having verified the high selectivity of probe 3 for sensing NTR activity in vitro, we studied the ability of the
probe to detect the NTR in living bacterial pathogens, containing two Gram-positive species, S. aureus and
Methicillin-resistant S. aureus (MRSA), and two Gram-negative species, K. pneumoniae and E. coli. First, probe 3
ACS Paragon Plus Environment

Page 13 of 34
The Journal of Organic Chemistry
1
2
3
4
and Cy 5 carboxyl acid (10 μM) (Figure S10) were incubated with four bacterial strains in Tris buffer (0.05 M, pH
5
6
7
o
7.4) at 37 C for 1 h, respectively. Then the nucleoid was stained with Hoechst 33258 and the bacteria were
8
9
imaged by confocal fluorescence microscopy. As shown in Figure 6a, c, e and g, all the four tested bacterial
strains incubated with Cy 5 control showed very weak fluorescence signals exciting at 633nm, indicating that Cy 5
carboxyl acid cannot penetrate the tested bacterial cells. However, all the bacteria exhibited bright fluorescent
signal after treated with probe 3, which demonstrated that probe 3 was readily taken up and activated by NTRs in
live bacterial cells. In addition, the quantification of fluorescence intensity in the bacteria revealed that
pre-treatment of bacterial cells with the inhibitor dicoumarin had a significant effect on the reduction of probe 3
(Figure 6b, d, f and h). The fluorescence intensity of these four bacterial strains decreased by 50 % to 60 % in the
presence of dicoumarin (Figure 6e), suggesting that the probe 3 was largely activated by NTR intracellularly after
internalization.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 34
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Confocal microscopy images of E. coli (a and b), S. aureus (c and d), MRSA (e and f) and K.
pneumoniae (g and h) incubated with probe 3 and Cy 5 carboxyl acid (10 μM) in the absence or presence of NTR
inhibitor (dicoumarin (0.1 mM)), respectively. DNA was stained with Hoechst 33258 (blue), red: probe and Cy 5
signals. (e) Quantification of fluorescence intensity of probe 3 incubated with E. coli, S. aureus, MRSA, and K.
pneumoniae in the absence (red bars) or presence (green bars) of NTR inhibitor, respectively. Bars represent mean
values ± SD (n = 5). Hoechst signal (blue): λex = 405 nm and λem = 460 ± 30 nm; probe and Cy 5 signal (red):
λex = 633 nm and λem = 700 ± 50 nm. Scale bar = 10 μm.
Conclusion
Live-cell fluorescence microscopy of bacteria has allowed researchers to understand many fundamental
biological processes. The low permeability of the bacterial cell wall and low target-selectivity have been
identified as two major challenging barriers that prevent the visualization of the molecular events inside live
bacteria by fluorescent probes. Herein, we present the design and synthesis of two novel series of
NTR-triggered fluorogenic probes based on Cy 5 with various lipophilic nitroaromatic moieties as caging
groups decorating at the benzene unit or at the methine backbone of Cy 5 core. The introduction of lipophilic
nitroaromatic moieties make the cell-impermeable Cy 5 could penetrate both Gram-positive and
Gram-negative bacterial cell envelopes. The selected fluorogenic probe 3 demonstrates favorable
ACS Paragon Plus Environment

Page 15 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
characteristics in terms of high specificity, high sensitivity, rapid response to NTR over other biological
analytes, thus minimizing the background signal and enabling the real-time intracellular identification and
imaging of NTR in live bacterial cells.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental Section
General methods
All the chemicals were purchased from J&K, Energy Chemical or Innochem. Commercially available
reagents were used without further purification. Nitroreductase (≥100 units/mg) from Escherichia coli and
reduced nicotinamide adenine dinucleotide (NADH) were purchased from Sigma-Aldrich. Four bacterial
strains (Escherichia coli (E. coli) (ATCC 25922), Staphylococcus aureus (S. aureus) (ATCC 29213),
Klebsiella pneumoniae (K. pneumoniae) (ATCC 700603), Methicillin resistant Staphylococcus aureus
(MRSA) (ATCC 33592)) were purchased from American Type Culture Collection (ATCC), USA.
Fluorescence emission spectra and full wavelength absorption spectra were performed on Tecan Spark™
10M Multimode Microplate Reader. OD values were recorded in a 10 mm path quartz cell on a Metash
UV-5100B spectrometer. Confocal laser scanning microscope imaging were conducted with ZEISS LSM
710 Confocal Microscope. All 1H NMR spectra were recorded at 400 MHz. 13C NMR spectra
(proton-decoupled carbon data) were recorded at 100 MHz, 125 MHz or 150 MHz, respectively. HRMS was
measured with Thermo LCQ Deca XP Max mass spectrometer for ESI and Ion trap.
The Reaction and NMR Data
2,3,3-trimethyl-5-iodo-3H-indole (9c). The 4-iodine phenylhydrazine (511 mg ， 2.18 mmol) and
3-methyl-2-butanone (320 mg, 3.72 mmol) were added to the EtOH (20 mL) and added H2SO4 (32 μL), the
mixture was stirred and refluxed for 4 h. The solution was cooled down to rt and added Na2CO3 (pH = 7.0).
The mixture was poured into water (20 mL), and extracted with DCM (2 × 20 mL). The combined organic
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 34
1
2
3
4
5
6
7
8
9
layer was washed with water (2 × 20 mL), and dried over Na2SO4. The solvent was evaporated under
reduced pressure to give red solid 607 mg, yield 97 %. Mp 130 – 132 °C. 1H NMR (400 MHz, DMSO-d6) δ
7.81 (d, J = 2.0 Hz, H, -Ar), 7.61 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, H, -Ar), 7.24 (d, J = 8.0 Hz, H, -Ar), 2.20 –
2.16 (m, 3H, -CH3), 1.23 (s, 6H, -CH3). 13C NMR (125 MHz, DMSO-d6) δ 189.0, 153.7, 149.2, 136.6, 131.1,
120.0, 90.7, 54.2, 22.7, 15.5. HRMS (m/z) (M+H): calcd. for C11H13NI 286.0087, found 286.0079.
1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium bromide (10a). The compound 9a (10 g, 62.89 mmol)
and 6-bromo hexanic acid (18 g, 92.31 mmol) were added to the o-dichlorobenzen (50 mL) and stirred at
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
120 C for 24 h. After the reaction was complete, the solution was cooled down to rt and the product was
1
washed by ether. After high-temperature drying to give red solid 14 g, yield 63 %. Mp 132 – 134 °C. H
NMR (400 MHz, DMSO-d6) δ 8.02 – 8.00 (m, H, -Ar), 7.88 – 7.85 (m, H, -Ar), 7.62 – 7.60 (m, 2H, -Ar),
4.49 (t, J = 7.6 Hz, 2H, -CH2-), 2.88 (s, 3H, -CH3), 2.22 (t, J = 6.8 Hz, 2H, -CH2-), 1.89 – 1.81 (m, 2H,
-CH2-), 1.58 – 1.51 (m, 8H, -CH2-, -CH3), 1.47 – 1.41 (m, 2H, -CH2-). 13C NMR (125 MHz, DMSO-d6) δ
197.1, 174.8, 142.4, 141.6, 129.9, 129.4, 124.1, 116.1, 54.7, 48.1, 34.0, 27.5, 26.0, 24.6, 22.6, 14.8. HRMS
(m/z) (M+): calcd. for C17H24O2N+ 274.1802, found 274.1807.
1,2,3,3-tetramethyl-5-nitro-3H-indolium iodide (10b). A mixture of compound 9b (100 mg, 0.49 mmol)
and CH3I (105 mg, 0.74 mmol) was stirred with acetonitrile (3 mL) in sealing tube at 95 °C for 12 h and the
solution was cooled down to rt and the solvent was evaporated under reduced pressure. Then the product was
1
washed by ether, after high-temperature drying to give red solid 89 mg, yield 51 %. Mp 120 – 122 °C. H
NMR (400 MHz, Methanol-d4) δ 8.70 (s, 1H, -Ar), 8.55 (d, J = 6.4 Hz, 1H, -Ar), 8.11 (d, J = 6.8 Hz, 1H,
-Ar), 4.14 (s, 3H, -CH3), 1.70 (s, 9H, -CH3). HRMS (m/z) (M+): calcd. for C12H15O2N2+ 219.1128, found
219.1130.
1,2,3,3-tetramethyl-5-iodo-3H-indolium iodide (10c). Following the same procedure for synthesis of
ACS Paragon Plus Environment

Page 17 of 34
The Journal of Organic Chemistry
1
2
3
4
compound 10b and using compound 9c (330mg, 1.16 mmol), 10c was synthesized. The product was washed
5
6
7
1
by ether, after high-temperature drying to give red solid 442 mg, yield 81 %. Mp 108 – 110 °C. H NMR
8
9
(400 MHz, DMSO-d6) δ 8.29 (s, H, -Ar), 7.99 (d, J = 7.6 Hz, H, -Ar), 7.72 (d, J = 7.6 Hz, H, -Ar), 3.94 (s,
3H, -CH3), 2.75 (s, 3H, -CH3), 1.52 (s, 6H, -CH3). 13C NMR (125 MHz, DMSO-d6) δ 189.0, 153.7, 149.2,
136.6, 131.1, 122.0, 90.7, 54.2, 22.7, 15.5. HRMS (m/z) (M+): calcd. for C12H15NI+ 300.0244, found
300.0245.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1-ethyl-2,3,3-trimethyl-3H-indolium bromide (10d). Following the same procedure for synthesis of
compound 10a and using compound 9a (10 g, 62.89 mmol) and bromine ethane (13.7 g, 12.57 mmol), 10d
was synthesized. After high-temperature drying to give light purple solid 13 g, yield 77 %. Mp 120 – 122 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.03 – 8.00 (m, H, -Ar), 7.88 – 7.86 (m, H, -Ar), 7.62 – 7.60 (m, 2H, -Ar),
4.56 – 4.51 (m, 2H, -CH2-), 2.88 (s, 3H, -CH3), 1.54 (s, 6H, -CH3), 1.45 (t, J = 7.6 Hz, 3H, -CH3). 13C NMR
(100 MHz, DMSO-d6) δ 196.7, 142.5, 141.3, 129.9, 129.5, 124.1, 115.9, 54.7, 43.7, 22.5, 14.7, 13.3. HRMS
(m/z) (M+): calcd. for C13H18N+ 188.1434, found 188.1441.
N-((1E, 2E)-3-(phenylamino)allylidene)benzenaminium chloride (11). Tetramethoxypropane (4.9 g,
29.88 mmol) and hydrochloric acid (4.25 mL) were added to distilled water (90 mL), and stirred at 50 °C.
Then the solution of aniline (5.6 g, 60.22 mmol), hydrochloric acid (5.0 mL) and distilled water (70 mL) was
added dropwise to the reaction and continued to stir at 50 °C for 2 h. After cooling, filtered, dried and got
solid 5.63 g, yield 73 %. Mp 118 – 120 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.74 (s, 2H, -NH-), 8.98 (d, J
= 11.2 Hz, 2H, -Ar), 7.45 (d, J = 4.4 Hz, 8H, -Ar), 7.25 – 7.21 (m, 2H, -CH-), 6.54 (t, J = 11.6 Hz, 1H,
-CH-). 13C NMR (125 MHz, DMSO-d6) δ 159.0, 139.2, 130.3, 126.3, 117.9, 99.2. HRMS (m/z) (M+): calcd.
for C15H15N2+ 223.1230, found 223.1240.
N-((1E, 2Z)-2-bromo-3-(phenylamino)allylidene)benzenaminium bromide (12). The aniline (0.73 g, 7.84
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 34
1
2
3
4
5
6
7
8
9
mmol) was added to EtOH (20 mL) and stirred. The dibromo-4-oxobut-2-enoic acid (1.0 g, 3.89 mmol) was
added to EtOH (20 mL), and were added dropwise to the solution of aniline at 0 °C. Then continued to stir at
60 °C for 4 h. After cooling, filtered, dried and got solid 0.95 g, yield 81 %. Mp 135 – 137 °C. 1H NMR (400
MHz, DMSO-d6) δ 9.51 (s, 2H, -Ar), 7.69 (d, J = 8.0 Hz, 4H, -Ar), 7.50 (t, J = 8.0 Hz, 4H, -Ar), 7.36 – 7.31
(m, 2H, -CH-). 13C NMR (100 MHz, DMSO-d6) δ 158.1, 139.1, 132.6, 130.3, 128.3, 127.5, 123.4, 120.1,
89.8. HRMS (m/z) (M+): calcd. for C15H14BrN2+ 301.0335, found 301.0344.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-((1E, 3E)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-iodo-1,3,3-
trimethyl-3H-indolium iodide (13). The compound 11 (1.2 g, 4.64 mmol) and compound 10a (1.6 g, 4.52
mmol) were added to the acetic (10 mL) acetic anhydride (10 mL) and the mixture was stirred and refluxed
for 2 h. The solution was cooled down to rt and the solvent was evaporated under reduced pressure. Then
o
acetic (10 mL), pyridine (10 mL) and compound 10c (2 g, 4.68 mmol) were stirred at 120 C for 2 h. After
the reaction was complete, the product was purified by silica gel column chromatography (DCM : MeOH =
20 : 1) to give deep blue solid 2.1 g, yield 61 %. Mp 156 – 157 °C. 1H NMR (400 MHz, Methanol-d4) δ 8.32
– 8.18 (m, 2H, -CH-), 7.77 (d, J = 1.2 Hz, 1H, -Ar), 7.71 – 7.68 (m, 1H, -Ar), 7.51 (d, J = 7.2 Hz, 1H, -Ar),
7.43 (t, J = 8.4 Hz , 1H, -Ar), 7.35 (d, J = 8.0 Hz, 1H, -Ar), 7.29 (t, J = 7.2 Hz, 1H, -Ar), 7.05 (d, J = 8.4 Hz,
1H, -Ar), 6.68 (t, J = 12.4 Hz , 1H, -CH-), 6.39 (d, J = 13.6 Hz, 1H, -CH-), 6.20 (d, J = 13.6 Hz, 1H, -CH-),
4.17 (t, J = 7.2 Hz, 2H, -CH2-), 3.56 (s, 3H, -CH3), 2.32 (t, J = 7.2 Hz, 2H, -CH2-), 1.88 – 1.80 (m, 2H,
-CH2-), 1.73 (s, 6H, -CH3), 1.70 (s, 6H, -CH3), 1.67 – 1.65 (m, 2H, -CH2-), 1.55 – 1.49 (m, 2H, -CH2-). 13
C
NMR (150 MHz, Methanol-d4) δ 177.3, 175.9, 173.4, 156.3, 154.9, 148.3, 144.7, 144.4, 143.4, 143.0, 138.7,
132.4, 129.9, 127.2, 126.8, 126.4, 123.5, 113.4, 112.5, 105.5, 103.9, 88.4, 51.0, 50.1, 45.1, 34.6, 31.5, 30.8,
28.3, 27.9, 27.3, 25.7. HRMS (m/z) (M+): calcd. for C32H38O2N2I+ 609.1972, found 609.1959.
2-((1E, 3Z)-3-bromo-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1-
ACS Paragon Plus Environment

Page 19 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
ethyl-3,3-dimethyl-3H-indolium bromide (14). Compound 12 (500 mg, 1.31 mmol) and compound 10d
(320 mg, 1.20 mmol) were added to (Ac)2O (20 mL) and the mixture was stirred at 60 oC for 4 h. Then with
compound 10a (425 mg, 1.20 mmol), NaOAc (295 mg, 3.60 mmol), the mixture was stirred at 50 oC for 2h.
After the reaction was complete, the product was purified by silica gel column chromatography (DCM :
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
MeOH = 20 : 1) to give deep blue solid 437 mg, yield 51 %. Mp 135 – 137 °C. H NMR (400 MHz,
Methanol-d4) δ 8.43 (d, J = 13.2 Hz, 2H, -CH-), 7.57 (d, J = 6.8 Hz, 2H, -Ar), 7.49 – 7.35 (m, 4H, -Ar), 7.21
(d, J = 7.2 Hz, H, -Ar), 7.17 – 7.12 (m, H, -Ar), 6.50 (t, J = 12.8 Hz, 2H, -CH-), 4.30 – 4.20 (m, 4H, -CH2-),
2.34 (t, J = 6.8 Hz, 2H, -CH2-), 1.92 (t, J = 6.8 Hz, 2H, -CH2-), 1.78 (s, 12H, -CH3), 1.75 – 1.72 (m, 2H,
-CH2-), 1.59 – 1.54 (m, 2H, -CH2-), 1.47 (t, J = 6.8 Hz, 3H, -CH3). 13C NMR (150 MHz, Methanol-d4) δ
174.8, 174.5, 149.8, 149.6, 141.8, 141.6, 141.5, 141.4, 130.9, 128.5, 125.6, 122.2, 122.1, 115.6, 111.2,
111.0, 102.3, 101.9, 100.0, 100.0, 65.2, 49.6, 49.6, 43.8, 39.1, 33.3, 26.6, 26.0, 25.9, 24.2, 18.8, 12.6, 10.9.
HRMS (m/z) (M+): calcd. for C33H40O2N2Br+ 575.2268, found 575.2270.
(4-((((4-nitrobenzyl)oxy)carbonyl)amino)phenyl)boronic acid (15). The 4-nitrophenyl chloroformate (125
mg, 0.58 mmol), 4-amino phenyl boric acid (100 mg, 0.81 mmol) and NaHCO3 (145 mg, 1.73 mmol) were
added to the THF (5 mL) mixture was stirred at rt for 1 h and THF was evaporated to dryness. DCM was
added and the mixture was filtered and got white solid, the solid was washed with DCM (30 mL), dried and
1
got white solid 108 mg, yield 59 % (Scheme S1). Mp 155 – 156 °C. H NMR (400 MHz, Methanol-d4) δ
8.22 (d, J = 8.8 Hz, 2H, -Ar), 7.63 (d, J = 9.6 Hz, 2H, -Ar), 7.55 (d, J = 8.4 Hz, 2H, -Ar), 7.44 – 7.37 (m, 2H,
-Ar), 5.28 (s, 2H, -CH2-). 13C NMR (150 MHz, Methanol-d4) δ 155.2, 149.0, 145.9, 141.5, 135.8, 135.7,
129.3, 124.7, 118.8, 66.1, 49.5. HRMS (m/z) (M+H): calcd. for C14H13O6N2B 316.0867, found 316.0863.
(4-(((((4-nitrobenzyl)oxy)carbonyl)amino)methyl)phenyl)boronic acid (16). Following the same
procedure for synthesis of compound 15 and using the 4-nitrophenyl chloroformate (377 mg, 1.75 mmol)
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 34
1
2
3
4
and 4-(aminomethyl)phenylboronic acid (200 mg, 1.46 mmol), compound 16 was synthesized. The product
5
6
7
1
was purified by Pre-TLC (PE : EA = 3 : 1) to give white solid 53 mg, yield 11 % (Scheme S1). H NMR
8
9
(400 MHz, Methanol-d4) δ 8.21 (d, J = 8.8 Hz, 2H, - Ar), 7.70 (d, J = 9.2 Hz, 1H, -Ar), 7.59 – 7.56 (m, 3H,
-Ar), 7.29 – 7.23 (m, 2H, -Ar), 5.22 (s, 2H, -CH2-), 4.31 (s, 2H, -CH2-). 13C NMR (150 MHz, Methanol-d4) δ
157.1, 147.5, 144.7, 141.0, 140.5, 133.7, 133.4, 127.6, 126.1, 125.9, 123.1, 114.8, 64.7, 44.1. HRMS (m/z)
(M+H): calcd. for C15H16O6N2B 331.1096, found 331.1093.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(4-(((4-nitrobenzyl)oxy)carbonyl)phenyl)boronic acid (17). The 4-carboxyphenylboronic acid (500 mg,
3.01 mmol), 4-nitrobenzyl bromide (781 mg, 3.62 mmol) and NaHCO3 (380 mg, 4.52 mmol) were added to
the DMF (5 mL) mixture was stirred at rt for 2 h and DMF was evaporated to dryness. The product was
purified by silica gel column chromatography (PE : EA = 3 : 1) to give white solid 86 mg, yield 10 %
1
(Scheme S1). H NMR (400 MHz, Methanol-d4) δ 8.27 – 8.25 (m, 2H, -Ar), 8.04 (d, J = 6.8 Hz, 2H, -Ar),
7.73 – 7.69 (m, 4H, -Ar), 5.49 (s, 2H, -CH2-). 13C NMR (150 MHz, Methanol-d4) δ 166.1, 147.7, 143.7,
133.6, 133.1, 128.1, 123.3, 64.9. HRMS (m/z) (M+H): calcd. for C14H13O6NB 302.0752, found 302.0738.
2-((1E, 3E)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimet-
hyl-5-nitro-3H-indolium iodide (Probe 1). The compound 11 (150 mg, 0.58 mmol) and compound 10a
(205 mg, 0.58 mmol) were added to acetic (3 mL), acetic anhydride (3 mL) and the mixture was stirred and
refluxed for 2 h. The solution was cooled down to rt and the solvent was evaporated under reduced pressure.
Then acetic (3 mL), pyridine (3 mL) and compound 10b (200 mg, 0.58 mmol) were stirred at 120 oC for 2 h.
After the reaction was complete, the product was purified by silica gel column chromatography (DCM :
1
MeOH = 20 : 1) to give deep blue solid 160 mg, yield 42 %. Mp 158 – 160 °C. H NMR (400 MHz,
Methanol-d4) δ 8.43 – 8.37 (m, 1H, -CH-), 8.30 – 8.25 (m, 2H, -Ar), 8.22 (t, J = 13.2 Hz, 1H, -CH-), 7.61 (d,
J = 7.2 Hz, 1H, -Ar), 7.52 – 7.50 (m, 2H, -Ar), 7.44 – 7.40 (m, 1H, -Ar), 7.25 (d, J = 8.8 Hz, 1H, -Ar), 6.77
ACS Paragon Plus Environment

Page 21 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
(t, J = 12.0 Hz, 1H, -CH-), 6.65 (d, J = 14.4 Hz, 1H, -CH-), 6.19 (d, J = 12.8 Hz, 1H, -CH-), 4.30 (t, J = 7.2
Hz, 2H, -CH2-), 3.57 (s, 3H, -CH3), 2.32 (t, J = 7.2 Hz, 2H, -CH2-), 1.93 – 1.85 (m, 2H, -CH2-), 1.78 (s, 6H,
-CH3), 1.76 (s, 6H, -CH3), 1.72 – 1.66 (m, 2H, -CH2-), 1.56 – 1.48 (m, 2H, -CH2-). 13C NMR (150 MHz,
Methanol-d4) δ 179.0, 171.1, 157.8, 152.9, 150.2, 144.9, 143.8, 142.9, 130.1, 128.9, 128.3, 126.7, 123.8,
119.0, 113.8, 111.5, 110.1, 108.7, 104.2, 52.0, 46.0, 34.7, 31.1, 30.8, 28.7, 28.1, 27.5, 27.3, 25.7. HRMS
(m/z) (M+): calcd. for C32H38O4N3+ 528.2857, found 528.2863.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-((1E, 3E)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimet-
hyl-5-(4-((((4-nitrobenzyl)oxy)carbonyl)amino)phenyl)-3H-indolium iodide (Probe 2). A mixture of
compound 13 (50 mg, 0.07 mmol), compound 15 (26 mg, 0.08 mmol), Pd(dppf)2Cl2 (5 mg, 0.007 mmol) and
K2CO3 (28 mg, 0.20 mmol) was stirred in 1,4-dioxane (5 mL) and H2O (200 μL) at 90 oC for 1.5 h and then
the solvent was evaporated to dryness. The residue was purified by silica gel column chromatography
(DCM : MeOH = 20 : 1) to give dark blue solid 38 mg, yield 64 %. Mp 148 – 150 °C. 1H NMR (400 MHz,
Methanol-d4) δ 8.28 – 8.25 (m, 4H, -CH-, -Ar), 7.73 (d, J = 2.0 Hz, 1H, -Ar), 7.69 (t, J = 2.4 Hz, 1H, -Ar),
7.67 (d, J = 2.0 Hz, 1H, -Ar), 7.64 (d, J = 2.0 Hz, 1H, -Ar), 7.62 – 7.55 (m, 4H, -Ar), 7.50 – 7.48 (m, 1H,
-Ar), 7.43 – 7.39 (m, 1H, -Ar), 7.34 (d, J = 8.4 Hz, 1H, -Ar), 7.30 – 7.24 (m, 2H, -Ar), 6.64 (t, J = 12.4 Hz,
1H, -CH-), 6.31 – 6.27 (m, 2H, -CH-), 5.34 (s, 2H, -CH2-), 4.11 (t, J = 7.6 Hz, 2H, -CH2-), 3.65 (s, 3H,
-CH3), 2.32 (t, J = 7.2 Hz, 2H, -CH2-), 1.83 (t, J = 8.4 Hz, 2H, -CH2-), 1.78 (s, 6H, -CH3), 1.73 (s, 6H, -CH3),
1.70 – 1.66 (m, 2H, -CH2-), 1.54 – 1.46 (m, 2H, -CH2-). 13C NMR (150 MHz, Methanol-d4) δ 177.3, 175.1,
174.6, 155.2, 145.9, 143.6, 143.4, 142.7, 139.4, 132.4, 129.9, 129.8, 129.4, 128.4, 128.1, 126.8, 126.3,
124.7, 123.5, 121.5, 120.2, 112.1, 104.7, 104.4, 66.7, 66.2, 50.6, 48.4, 44.8, 34.7, 33.1, 31.8, 31.7, 30.8,
+
30.5, 28.2, 28.0, 27.9, 27.4, 25.7, 23.8, 20.3, 14.1. HRMS (m/z) (M+): calcd. for C46H49O6N4 753.3647,
found 753.3623.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 34
1
2
3
4
2-((1E, 3E)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimet-
5
6
7
hyl-5-(4-(((((4-nitrobenzyl)oxy)carbonyl)amino)methyl)phenyl)-3H-indolium
iodide
(Probe
3).
8
9
Following the same procedure for synthesis of probe 2 and using compound 13 (50 mg, 0.07 mmol) and
compound 16 (27 mg, 0.08 mmol), probe 3 was synthesized. The residue was purified by silica gel column
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
chromatography (DCM : MeOH = 20 : 1) to give dark blue solid 32 mg, yield 53 %. Mp 135 – 137 °C. H
NMR (400 MHz, Methanol-d4) δ 8.29 – 8.22 (m, 4H, -CH-, -Ar), 7.74 (s, 1H, -Ar), 7.67 (d, J = 8.0 Hz, 1H,
-Ar), 7.61 (d, J = 8.4 Hz, 4H, -Ar), 7.49 (d, J = 6.8 Hz, 1H, -Ar), 7.43 – 7.34 (m, 4H, -Ar), 7.31 – 7.25 (m,
2H, -Ar), 6.65 (t, J = 12.4 Hz, 1H, -CH-), 6.32 – 6.27 (m, 2H, -CH-), 5.25 (s, 2H, -CH2-), 4.36 (s, 2H,
-CH2-), 4.12 (t, J = 7.2 Hz, 2H, -CH2-), 3.65 (s, 3H, -CH3), 2.32 (t, J = 7.2 Hz, 2H, -CH2-), 1.88 – 1.82 (m,
2H, -CH2-), 1.78 (s, 6H, -CH3), 1.73 (s, 6H, -CH3), 1.70 – 1.66 (m, 2H, -CH2-), 1.54 – 1.44 (m, 2H, -CH2-).
13C NMR (150 MHz, Methanol-d4) δ 177.4, 175.1, 174.8, 158.6, 155.6, 155.3, 146.2, 143.7, 143.6, 143.4,
142.7, 140.6, 139.9, 139.4, 129.8, 129.2, 129.0, 128.5, 128.1, 126.8, 126.3, 124.6, 123.5, 121.8, 112.1,
104.6, 66.2, 50.7, 50.6, 45.3, 34.7, 31.7, 30.8, 30.5, 30.4, 28.2, 28.0, 27.4, 27.0, 25.7. HRMS (m/z) (M+):
calcd. for C47H51O6N4+ 767.3803, found 767.3780.
2-((1E, 3E)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimet-
hyl-5-(4-nitrophenyl)-3H-indolium iodide (Probe 4). Following the same procedure for synthesis of probe
2 and using compound 13 (50 mg, 0.07 mmol) and 4-nitrophenylboronic acid (13.6 mg, 0.08 mmol), probe 4
was synthesized. The residue was purified by silica gel column chromatography (DCM : MeOH = 20 : 1) to
give dark blue solid 45 mg, yield 71 %. Mp 138 – 140 °C. 1H NMR (400 MHz, Methanol-d4) δ 8.33 – 8.23
(m, 4H, -CH-, -Ar), 7.92 (t, J = 2.4 Hz, 1H, -Ar), 7.90 (t, J = 2.0 Hz, 1H, -Ar), 7.87 (d, J = 1.6 Hz, 1H, -Ar),
7.80 – 7.77 (m, 1H, -Ar), 7.53 – 7.51 (m, 1H, -Ar), 7.46 – 7.41 (m, 1H, -Ar), 7.39 – 7.35 (m, 2H, -Ar), 7.32
– 7.28 (m, 1H, -Ar), 6.68 (t, J = 12.4 Hz, 1H, -CH-), 6.39 (d, J = 14.0 Hz, 1H, -CH-), 6.26 (d, J = 13.6 Hz,
ACS Paragon Plus Environment

Page 23 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1H, -CH-), 4.17 (t, J = 7.6 Hz, 2H, -CH2-), 3.63 (s, 3H, -CH3), 2.32 (t, J = 7.2 Hz, 2H, -CH2-), 1.89 – 1.83
(m, 2H, -CH2-), 1.79 (s, 6H, -CH3), 1.74 (s, 6H, -CH3), 1.70 – 1.66 (m, 2H, -CH2-), 1.55 – 1.48 (m, 2H,
-CH2-). 13C NMR (150 MHz, Methanol-d4) δ 177.4, 175.9, 174.2, 156.3, 154.9, 148.3, 148.1, 145.3, 143.5,
143.4, 143.0, 136.5, 130.0, 129.2, 128.7, 127.2, 126.8, 125.2, 123.5, 122.4, 112.6, 112.0, 105.5, 104.3, 51.0,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
50.3, 45.1, 34.7, 31.6, 28.3, 28.0, 27.9, 27.4, 25.8. HRMS (m/z) (M+): calcd. for C38H42O4N3 604.3170,
found 604.3175.
2-((1E, 3Z)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)-3-(4-nitrophenyl)penta-1,3-dien-
1-yl)-1-ethyl-3,3-dimethyl-3H-indolium bromide (Probe 5). Following the same procedure for synthesis
of probe 2 and using compound 14 (100 mg, 0.15 mmol), 4-nitrophenylboronic acid (31 mg, 0.18 mmol),
probe 5 was synthesized. The residue was purified by silica gel column chromatography (DCM : MeOH =
20: 1) to give dark blue solid 56 mg, yield 53 %. Mp 130 – 132 °C. 1H NMR (400 MHz, Methanol-d4) δ 8.46
(d, J = 8.0 Hz, 2H, -Ar), 8.39 (d, J = 14.0 Hz, 2H, -CH-), 7.59 (d, J = 8.0 Hz, 2H, -Ar), 7.52 – 7.49 (m, 2H,
-Ar), 7.40 – 7.36 (m, 2H, -Ar), 7.29 – 7.25 (m, 4H, -Ar), 5.68 – 5.62 (m, 2H, -CH-), 3.86 – 3.77 (m, 4H,
-CH2-), 2.18 (t, J = 7.2 Hz, 2H, -CH2-), 1.78 (s, 12H, -CH3), 1.62 – 1.58 (m, 2H, -CH2-), 1.47 – 1.42 (m, 2H,
-CH2-), 1.26 (s, 2H, -CH2-), 1.16 (t, J = 7.2 Hz, 3H, -CH3). 13C NMR (150 MHz, Methanol-d4) δ 177.7,
175.3, 175.0, 153.8, 149.1, 144.7, 143.3, 142.9, 142.8, 133.0, 130.9, 129.9, 126.8, 125.7, 123.5, 112.3,
112.1, 102.2, 101.6, 71.6, 50.9, 50.8, 44.9, 40.1, 34.9, 30.8, 28.0, 27.8, 27.7, 27.4, 25.6, 12.3. HRMS (m/z)
(M+): calcd. for C39H44O4N3+ 618.3326, found 618.3315.
2-((1E, 3Z)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)-3-(4-((((4-nitrobenzyl)oxy)carb-
onyl)amino)phenyl)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indolium
bromide
(Probe
6).
Following the same procedure for synthesis of probe 2 and using compound 14 (100 mg, 0.15 mmol),
compound 15 (58 mg, 0.18 mmol), probe 6 was synthesized. The residue was purified by silica gel column
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 34
1
2
3
4
5
6
7
8
9
1
chromatography (DCM : MeOH = 20 : 1) to give dark blue solid 59 mg, yield 50 %. Mp 160 – 162 °C. H
NMR (400 MHz, Methanol-d4) δ 8.33 (d, J = 14.0 Hz, 2H, -CH-), 8.25 (d, J = 8.8 Hz, 2H, -Ar), 7.71 – 7.67
(m, 4H, -Ar), 7.49 – 7.47 (m, 2H, -Ar), 7.39 – 7.34 (m, 2H, -Ar), 7.26 – 7.21 (m, 6H, -Ar), 5.81 – 5.73 (m,
2H, -CH-), 5.34 (s, 2H, -CH2-), 3.82 (d, J = 6.8 Hz, 2H, -CH2-), 3.74 (t, J = 6.8 Hz, 2H, -CH2-), 2.20 (t, J =
7.2 Hz, 2H, -CH2-), 1.75 (s, 12H, -CH3), 1.59 – 1.54 (m, 2H, -CH2-), 1.50 – 1.41 (m, 2H, -CH2-), 1.26 – 1.22
(m, 2H, -CH2-), 1.18 – 1.14 (m, 3H, -CH3). 13C NMR (150 MHz, Methanol-d4) δ 173.1, 172.9, 153.8, 153.1,
153.0, 147.6, 144.4, 141.9, 141.5, 141.3, 141.2, 138.7, 134.9, 130.5, 128.3, 128.0, 125.0, 125.0, 123.2,
122.1, 122.02, 119.0, 110.5, 110.4, 101.4, 100.8, 64.8, 49.2, 49.1, 43.4, 38.5, 33.8, 29.3, 26.5, 26.3, 26.1,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
24.3, 10.8. HRMS (m/z) (M+): calcd. for C47H51O6N4 767.3803, found 767.3802.
2-((1E, 3Z)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)-3-(4-(((((4-nitrobenzyl)oxy)carb-
onyl)amino)methyl)phenyl)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indolium bromide (Probe 7).
Following the same procedure for synthesis of probe 2 and using compound 14 (100 mg, 0.15 mmol),
compound 16 (60 mg, 0.18 mmol), probe 7 was synthesized. The residue was purified by silica gel column
1
chromatography (DCM : MeOH = 20 : 1) to give dark blue solid 36 mg, yield 54 %. Mp 132 – 134 °C. H
NMR (400 MHz, Methanol-d4) δ 8.33 (d, J = 14.0 Hz, 2H, -CH-), 8.21 (d, J = 8.4 Hz, 2H, -Ar), 7.62 (d, J =
8.4 Hz, 2H, -Ar), 7.52 (d, J = 8.0 Hz, 2H, -Ar), 7.48 (d, J = 7.2 Hz, 2H, -Ar), 7.38 – 7.35 (m, 2H, -Ar), 7.25
(d, J = 7.6 Hz, 4H, -Ar), 7.21 (d, J = 7.6 Hz, 2H, -Ar), 5.73 (dd, J1 = 14.0 Hz, J2 = 5.2 Hz, 2H, -CH-), 5.26 (s,
2H, -CH2-), 4.43 (s, 2H, -CH2-), 3.81 – 3.68 (m, 4H, -CH2-), 2.21 (t, J = 6.8 Hz, 2H, -CH2-), 1.75 (s, 12H,
-CH3), 1.58 – 1.55 (m, 2H, -CH2-), 1.50 – 1.46 (m, 2H, -CH2-), 1.22 – 1.17 (m, 2H, -CH2-), 1.12 (t, J = 6.8
Hz, 3H, -CH3). 13C NMR (150 MHz, Methanol-d4) δ 173.2, 172.9, 157.2, 152.9, 147.5, 144.8, 141.9, 141.5,
141.3, 141.2, 139.4, 134.8, 134.4, 130.1, 128.3, 127.7, 125.0, 123.2, 122.0, 110.6, 110.4, 101.4, 100.8, 64.8,
49.2, 49.1, 43.9, 43.4, 38.4, 33.6, 29.3, 26.5, 26.4, 26.3, 26.0, 24.2, 10.8. HRMS (m/z) (M+): calcd. for
ACS Paragon Plus Environment

Page 25 of 34
The Journal of Organic Chemistry
1
2
3
4
C48H53O6N4+ 781.3960, found 781.3966.
5
6
7
8
9
2-((1E, 3Z)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)-3-(4-(((4-nitrobenzyl)oxy)carb-
onyl)phenyl)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indolium bromide (Probe 8). Following the
same procedure for synthesis of probe 2 and using compound 14 (100 mg, 0.15 mmol), compound 17 (55
mg, 0.18 mmol), probe 8 was synthesized. The residue was purified by silica gel column chromatography
(DCM : MeOH = 20 : 1) to give dark blue solid 38 mg, yield 30 %. Mp 124 – 126 °C. 1H NMR (400 MHz,
Methanol-d4) δ 8.37 (dd, J1 = 14.0 Hz, J2 = 2.4 Hz, 2H, -CH-), 8.31 – 8.27 (m, 4H, - Ar), 7.76 (d, J = 8.4 Hz,
2H, -Ar), 7.51 – 7.46 (m, 4H, -Ar), 7.38 – 7.35 (m, 2H, -Ar), 7.28 – 7.22 (m, 4H, -Ar), 5.71 – 5.63 (dd, J1 =
18.8 Hz, J2 = 14.8 Hz, 2H, -CH-), 5.54 (s, 2H, -CH2-), 3.84 – 3.72 (m, 4H, -CH2-), 2.13 (t, J = 6.4 Hz, 2H,
-CH2-), 1.78 (s, 12H, -CH3), 1.58 – 1.54 (m, 2H, -CH2-), 1.43 – 1.39 (m, 2H, -CH2-), 1.29 – 1.26 (m, 2H,
-CH2-), 1.14 (t, J = 7.2 Hz, 3H, -CH3). 13C NMR (150 MHz, Methanol-d4) δ 175.0, 174.7, 167.1, 153.8,
149.3, 145.1, 143.3, 143.0, 142.9, 142.9, 142.7, 134.8, 133.0, 132.4, 132.0, 131.8, 130.9, 130.8, 130.1,
129.8, 126.7, 126.7, 124.8, 123.6, 123.5, 112.2, 112.0, 102.5, 101.9, 66.8, 50.8, 50.7, 44.9, 40.0, 35.1, 30.8,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
28.0, 27.8, 27.7, 27.5, 25.6, 20.3, 12.3. HRMS (m/z) (M+): calcd. for C47H50O6N3 752.3694, found
752.3680.
5-amino-2-((1E,3E)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,
3-trimethyl-3H-indol-1-ium iodide (Product 18). The probe 1 (100 mg, 0.15 mmol), Zn (100 mg, 1.52
mmol) and NH4Cl (82 mg, 1.52 mmol) were added to the THF (5 mL) and H2O (1 mL) mixture was stirred at
rt for 1 h, THF and H2O was evaporated to dryness. The product was purified by silica gel column
chromatography (DCM : MeOH = 10 : 1) to give dark blue solid 36 mg, yield 38 % (Scheme S2). Mp 142 –
144 °C. 1H NMR (500 MHz, Methanol-d4) δ 8.31 – 8.18 (m, 2H, -CH-), 7.53 (d, J = 6.5 Hz, H, -Ar), 7.46 –
7.43 (m, H, -Ar), 7.40 – 7.32 (m, 5H, -Ar), 6.65 (t, J = 12.0 Hz, H, -CH-), 6.40 (d, J = 14.0 Hz, H, -CH-),
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 34
1
2
3
4
5
6
7
8
9
6.20 (d, J = 13.0 Hz, H, -CH-), 4.17 (t, J = 7.5 Hz, 2H, -CH2-), 3.57 (s, 3H, -CH3), 2.32 (t, J = 6.0 Hz, 2H,
-CH2-), 2.03 (s, 2H, -CH2-), 1.85 (t, J = 8.0 Hz, 2H, -CH2-), 1.74 (s, 12H, -CH3), 1.51 (s, 2H, -CH2-). 13C
NMR (150 MHz, Methanol-d4) δ 177.9, 174.0, 156.7, 155.1, 144.8, 144.0, 143.6, 130.5, 127.5, 124.1, 123.7,
117.7, 113.2, 113.0, 104.7, 51.6, 50.8, 45.8, 35.2, 32.1, 28.9, 28.5, 28.4, 28.0, 26.3. HRMS (m/z) (M+): calcd.
for C32H40O2N3+ 498.3115, found 498.3115.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-(4-aminophenyl)-2-((1E,3E)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien
-1-yl)-1,3,3-trimethyl-3H-indol-1-ium iodide(Product 19). Following the same procedure for synthesis of
probe 2 and using compound 13 (100 mg, 0.14 mmol), (4-aminophenyl)boronic acid hydrochloride (28 mg,
0.16 mmol), product 19 was synthesized. The residue was purified by silica gel column chromatography
(DCM : MeOH = 10 : 1) to give dark blue solid 42 mg, yield 44 %. Mp 140 – 142 °C. 1H NMR (500 MHz,
Methanol-d4) δ 8.27 (dd, J1 = 24.5 Hz, J2 = 12.0 Hz, 2H, -CH-), 7.70 (s, 1H, -Ar), 7.63 (d, J = 8.0 Hz, 1H,
-Ar), 7.52 (d, J = 7.0 Hz, 1H, -Ar), 7.47 – 7.42 (m, 3H, -Ar), 7.36 (d, J = 8.5 Hz, 1H, -Ar), 7.31 – 7.27 (m,
2H, -Ar), 6.85 (d, J = 8.0 Hz, 2H, -Ar), 6.67 (t, J = 12.0 Hz, 1H, -CH-), 6.36 – 6.28 (dd, J1 = 26.0 Hz, J2 =
13.5 Hz, 2H, -CH-), 4.13 (t, J = 7.0 Hz, 2H, -CH2-), 3.70 (s, 3H, -CH3), 2.36 (s, 2H, -CH2-), 1.87 (t, J = 7.5
Hz, 2H, -CH2-), 1.81 (s, 6H, -CH3), 1.77 (s, 6H, -CH3), 1.73 (t, J = 7.0 Hz, 2H, -CH2-), 1.58 – 1.53 (m, 2H,
-CH2-). 13C NMR (150 MHz, Methanol-d4) δ 175.3, 174.0, 155.2, 154.8, 148.7, 143.7, 143.4, 142.5, 140.6,
131.2, 129.7, 128.7, 127.4, 126.7, 126.0, 123.4, 120.9, 116.9, 112.2, 111.8, 105.0, 104.0, 50.7, 50.4, 44.7,
31.9, 28.2, 28.1, 27.9, 27.4, 25.9. HRMS (m/z) (M+): calcd. for C38H44O2N3+ 574.3428, found 574.3428.
5-(4-(aminomethyl)phenyl)-2-((1E,3E)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta
-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium iodide(Product 20). Following the same procedure for
synthesis of probe 2 and using compound 13 (100 mg, 0.14 mmol), (4-(aminomethyl)phenyl)boronic acid
hydrochloride (31 mg, 0.16 mmol), product 20 was synthesized. The residue was purified by silica gel
ACS Paragon Plus Environment

Page 27 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
column chromatography (DCM : MeOH = 10 : 1) to give dark blue solid 18 mg, yield 19 %. Mp 160 – 162
°C. 1H NMR (500 MHz, Methanol-d4) δ 8.35 – 8.28 (m, 2H, -CH-), 7.82 – 7.80 (m, 3H, -Ar), 7.75 (d, J = 8.0
Hz, 1H, -Ar), 7.60 (d, J = 7.5 Hz, 2H, -Ar), 7.54 (t, J = 7.5 Hz, 1H, -Ar), 7.48 – 7.45 (m, 1H, -Ar), 7.41 (d, J
= 8.0 Hz, 1H, -Ar), 7.38 – 7.31 (m, 2H, -Ar), 6.70 (t, J = 12.5 Hz, 1H, -CH-), 6.39 – 6.31 (m, 2H, -CH-),
4.22 (s, 2H, -CH2-), 4.18 (t, J = 7.0 Hz, 2H, -CH2-), 3.69 (s, 3H, -CH3), 2.37 (t, J = 7.0 Hz, 2H, -CH2-), 1.89
(t, J = 7.5 Hz, 2H, -CH2-), 1.83 (s, 6H, -CH3), 1.78 (s, 6H, -CH3), 1.73 (t, J = 7.0 Hz, 2H, -CH2-), 1.55 (t, J =
6.5 Hz, 2H, -CH2-), 1.34 (d, J = 5.0 Hz, 2H, -CH2-). 13C NMR (150 MHz, Methanol-d4) δ 177.3, 175.3,
174.8, 155.8, 155.10, 144.2, 143.5, 143.4, 142.8, 142.6, 138.5, 133.5, 130.7, 129.8, 128.6, 127.0, 126.5,
123.5, 121.9, 112.3, 112.1, 104.9, 104.4, 50.8, 50.5, 45.0, 44.1, 34.6, 31.6, 28.3, 28.0, 27.9, 27.4, 25.7.
HRMS (m/z) (M+): calcd. for C39H46O2N3+ 588.3585, found 588.3588.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-((1E,3Z)-3-(4-aminophenyl)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien
-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium bromide(Product 21). Following the same procedure for
synthesis of probe 2 and using compound 14 (100 mg, 0.15 mmol), (4-aminophenyl)boronic acid
hydrochloride (34 mg, 0.18 mmol), product 21 was synthesized. The residue was purified by silica gel
column chromatography (DCM : MeOH = 10 : 1) to give dark blue solid 30 mg, yield 30 %. Mp 138 – 140
1
°C. H NMR (500 MHz, Methanol-d4) δ 8.34 (d, J = 16.0 Hz, 2H, -CH-), 7.55 – 7.53 (m, 2H, -Ar), 7.45 –
7.41 (m, 2H, -Ar), 7.31 – 7.28 (m, 4H, -Ar), 7.06 (d, J = 8.5 Hz, 2H, -Ar), 6.97 (d, J = 8.0 Hz, 2H, -Ar), 5.94
(t, J = 15.0 Hz, 2H, -CH-), 3.93 – 3.89 (m, 2H, -CH2-), 3.83 (t, J = 7.5 Hz, 2H, -CH2-), 2.31 (t, J = 7.0 Hz,
2H, -CH2-), 1.81 (s, 12H, -CH3), 1.71 – 1.66 (m, 2H, -CH2-), 1.60 (t, J = 7.5 Hz, 2H, -CH2-), 1.34 (t, J = 7.5
Hz, 2H, -CH2-), 1.24 (t, J = 7.0 Hz, 3H, -CH3). 13C NMR (150 MHz, Methanol-d4) δ 174.3, 174.0, 154.9,
154.8, 150.6, 149.1, 143.5, 143.1, 142.8, 142.6, 141.6, 137.7, 132.0, 129.8, 126.3, 125.3, 123.5, 123.5,
116.8, 111.9, 111.7, 103.2, 102.6, 50.5, 50.5, 44.9, 39.9, 28.1, 27.8, 27.8, 27.7, 26.1, 12.3. HRMS (m/z)
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 34
1
2
3
4
(M+): calcd. for C39H46O2N3+ 588.3585, found 588.3579.
5
6
7
UV-vis absorption and fluorescence spectra of probe 3
8
9
The concentration of DMSO stock solution of probe 3 was diluted to 5 μM in 0.05 M Tris buffer (pH 7.4)
with 1.5% DMSO as co-solvent. The UV-visible spectra were recorded using a Tecan Spark™ 10M
Multimode Microplate Reader. Wavelength interval: 2.0 nm. Fluorescence spectroscopic studies of probe 3
were performed at the excitation wavelength of 620 nm. Wavelength interval: 2.0 nm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cyclic voltammetry
Cyclic voltammetry was performed using a CHI600A analyzer with a scan rate of 100 mV/s at room
temperature. The electrolytic cell was a conventional three-electrode cell with a glassy carbon working
electrode, an Ag/Ag+ (0.01 M AgNO3) as the reference electrode and Pt wire as the counter electrode. The
redox potentials were measured in CH3CN with 0.1 M of tetra-n-butylammonium hexafluorophosphate
(n-Bu4NPF6) as a supporting electrolyte. ΔGeT values were calculated from the Rehm-Weller equation: ΔGeT
= Eox - Ered - ΔE0,0 - wp (I), where Eox and Ered are oxidation and reduction potentials of electron donor and
acceptor, respectively, ΔE0,0 is the excited energy, and wp is the work term for the charge separation and
very small.15 We simplify equation I to equation II: ΔGeT = Eox - Ered - ΔE0,0.
Confocal imaging of bacteria treated with probe 3
E. coli, S. aureus, MRSA, and K. pneumoniae cells were cultured for 12 h in respective media at 37 °C.
Bacterial strains cultured overnight in respective solution were harvested and washed twice with Tris buffer
(pH 7.4). The washed cells were resuspended in Tris buffer (pH 7.4) with an OD600 of 0.5-0.7. Then 500 μL
aliquots were treated with 10 μM of probe 3 or Cy 5 in the presence or absence of the enzyme inhibitor,
dicoumarin (0.1 mM). After incubation at 37 °C for 1 h, the cells were washed with Tris buffer (pH 7.4) by
centrifugation to remove the unbound reagents and then treated with 20 μg/mL of Hoechst 33258 at 37 °C
ACS Paragon Plus Environment

Page 29 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
for 30 min. Then a drop of the suspension was added into an 8-well chamber followed by covering with
agarose pads. Fluorescence images were acquired with ZEISS LSM 710 Confocal Microscope (Nikon
Eclipse TE2000-E, CFI PlanApochromat VC 63× oil immersion objective), using a high pressure He-Ne
lamp and diode laser for excitation. Hoechst 33258 was excited at 405 nm and its fluorescence was
monitored at 430-490 nm, while Cy 5 and probe 3 were excited at 633 nm and the fluorescence was
monitored at 650-750 nm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
1H NMR, 13C NMR spectra for all compounds and spectroscopic properties of probes.
ACKNOWLEDGMENTS
This work was partially supported by the National Natural Science Foundation of China (NSFC)
projects (21778077, 21502236), the Sino-German research project (GZ 1271), the Beijing Nova
Program (Z16111000490000), the CAMS Innovation Fund for Medical Sciences (CIFMS
2017-I2M-2-004) and the Fundamental Research Funds for the Central Universities (3332018150).
References
1. (a) Abalymov, A. A.; Verkhovskii, R. A.; Novoselova, M. V.; Parakhonskiy, B. V.; Gorin, D. A.;
Yashchenok, A. M.; Sukhorukov, G. B. Live-cell imaging by confocal raman and fluorescence
microscopy recognizes the crystal structure of calcium carbonate particles in heLa cells. Biotechnol. J.
2018, e1800071. (b) Sahl, S. J.; Hell, S. W.; Jakobs, S. Fluorescence nanoscopy in cell biology. Nat.
Rev. Mol. Cell. Biol. 2017, 18 (11), 685-701. (c) Schlafer, S.; Meyer, R. L. Confocal microscopy
imaging of the biofilm matrix. J. Microbiol. Methods. 2017, 138, 50-59. (d) Liu, J. N.; Bu, W.; Shi, J.
ACS Paragon Plus Environment