RETRACTED ARTICLE: Sanger's Reagent Sensitized Photocleavage of Amide Bond for Constructing Photocages and Regulation of Biological Functions

Article abstract of DOI:10.1021/jacs.9b11357

Photolabile groups offer promising tools to study biological processes with high spatial and temporal control. In the investigation, we designed and prepared several new glycine amide derivatives of Sanger's reagent and demonstrated that they serve as a new class of photocages for Zn²⁺ and an acetylcholinesterase (AChE) inhibitor. We showed that the mechanism for photocleavage of these substances involves initial light-driven cyclization between the 2,4-dinitrophenyl and glycine methylene groups to form acyl benzimidazole N-oxides, which undergo secondary photoinduced decarboxylation in association with rupture of an amide bond. The cleavage reactions proceed with modest to high quantum yields. We demonstrated that these derivatives can be used in targeted intracellular delivery of Zn²⁺, fluorescent imaging by light-triggered Zn²⁺ release, and regulation of biological processes including the enzymatic activity of carbonic anhydrase (CA), negative regulation of N-methyl-d-aspartate receptors (NMDARs), and pulse rate of cardiomyocytes. The successful proof-of-concept examples described above open a new avenue for using Sanger's reagent-based glycine amides as photocages for the exploration of complex cellular functions and signaling pathways.

Full text of DOI:10.1021/jacs.9b11357

Subscriber access provided by Murdoch University Library
Article
Sanger's Reagent Sensitized Photocleavage of Amide Bond for
Constructing Photocages and Regulation of Biological Functions
Tingwen Wei, sheng lu, Jiahui Sun, Zhijun Xu, Xiao Yang,
Fang Wang, Yang Ma, Yun Shi, and Xiaoqiang Chen
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b11357 • Publication Date (Web): 05 Feb 2020
Downloaded from pubs.acs.org on February 6, 2020
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 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Sanger's Reagent Sensitized Photocleavage of Amide Bond for
Constructing Photocages and Regulation of Biological Functions
Tingwen Wei1, Sheng Lu1, Jiahui Sun2,3, Zhijun Xu1, Xiao Yang1, Fang Wang1, Yang Ma1, Yun Stone
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
Shi*2,3,4 and Xiaoqiang Chen* 1
1State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering,
Nanjing Tech University, Nanjing 210009, China.
2State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210032, China.
3Ministry of Education Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing
University, Nanjing 210032, China.
4Chemistry and Biomedicine Innovation Center, Nanjing University, Nanjing 210032, China.
ABSTRACT: Photolabile groups offer promising tools to study biological processes with highly spatial and temporal control.
In the investigation, we designed and prepared several new glycine amide derivatives of Sanger’s reagent and demonstrated
that they serve as a new class of photocages for Zn2+ and an acetylcholinesterase (AChE) inhibitor. We showed that the
mechanism for photocleavage of these substances involves initial light-driven cyclization between the 2,4-dinitrophenyl and
glycine methylene groups to form acyl benzimidazole N-oxides, which undergo secondary photoinduced decarboxylation in
association with rupture of an amide bond. The cleavage reactions proceed with modest to high quantum yields. We
demonstrated that these derivatives can be used in targeted intracellular delivery of Zn2+, fluorescent imaging by light-
triggered Zn2+ release, and regulation of biological processes including the enzymatic activity of carbonic anhydrase (CA),
negative regulation of N-methyl-D-aspartate receptors (NMDARs) and pulse rate of cardiomyocytes. The successful proof-of-
concept examples described above open a new avenue for using Sanger’s reagent-based glycine amides as photocages for the
exploration of complex cellular functions and signaling pathways.
Despite their wide range of applications, ortho-
nitrobenzyl photocages often suffer from low quantum
INTRODUCTION
Light promoted bond cleavage reactions of photocaged
compounds serve as powerful tools to create materials in a
highly spatially and temporally controlled manner. As a
result, these substances have found wide applications in
tissue engineering, bio-sensing, counterfeit detection,
controlled release of functional molecules and regulation of
gene expression.1-8 Moreover, owing to their use of non-
invasive optical triggers, and its high spatial and temporal
precision, photocaged substances have been utilized to
explore biological processes and elucidate their
mechanistic nature.9-12 Arylcarbonylmethyl,13 nitroaryl
(Ortho-nitrobenzyl, Ortho-nitroanilides)14, azobenzene,15
xanthene,16 coumarin-4-ylmethyl,17-20 and indoline
groups21-22 are the commonly used photoreaction triggers
to construct photocages for controlled release of metal ions
(Ca2+, Zn2+, Cu2+), drugs and other bio-active substances. 23-
yields (<0.05) as a result of resonance stabilization of aci-
nitro intermediates produced by irradiation.14, 33 Recently,
Prem et al. demonstrated that novel photocaged
compounds, containing m-nitrophenyl acetic acid and
xanthone acetic acid chromophores, undergo irradiation
promoted decarboxylative C-C bond cleavage with quantum
yields up to 0.30.34 However, developing photocages that
are efficiently cleaved in photochemical reactions that
operate by new types of mechanistic pathways is still the
current challenge and interest.
Sanger’s reagent (2,4-dinitrofluorobenzene) (Scheme 1)
has been widely used for sequencing proteins since the
early 1940s.35-37 This well-studied reagent reacts with an
NH2 group of the N-terminal amino acid in a polypeptide.
Acid promoted cleavage of the resulting functionalized
polypeptide produces a unique N-dinitrophenylamino acid
that arises from the polypeptides N–terminal amino acid. 38-
40 Thoughts about the potential of utilizing Sanger’s reagent
to construct photocleavable substances aroused our recent
interest. In the study described below, we assessed this
proposal by preparing a series of glycine derivatives of
Sanger’s reagent, and determining the nature and
efficiencies of their photochemical reactions. We observed
that irradiation of these substances produce N-2,4-
27
Particularly, incorporation of nitrobenzene derivatives
along with metal ion-binding ligands has become
a
prevailing tool to investigate the signaling processes in
biological systems that are governed by metal ions. 28-31In
addition, photocage containing metal ion complexes along
with fluorophores whose optical properties are altered by
metal ion binding and release have been employed to
elucidate cellular processes and signaling.32
dinitrobenzimadazole N-oxides via
a mechanism that
ACS Paragon Plus Environment

Journal of the American Chemical Society
differs from that of photocleavage reactions of conventional
Page 2 of 9
(m/z=145.0740) are generated in photoreaction of the
Sanger’s reagent derived amide 2 (Figures 1a, S2-S10).
HPLC was used to monitor and quantify the decomposition
products generated from the photoreaction of compound 2.
During the decomposition of compound 2, the content of 2-
G1 increases within the first 30 sec then drops, indicating a
consumption of 2-G1; while the contents of 2-G2 and 2-G4
elevate throughout (Figure S11). Hence, we hypothesize
that 2-G2 and 2-G4 are the products of a secondary
photoreaction of 2-G1. To evidence this, we further
investigated the photoreaction of isolated 2-G1. Upon UV
irradiation, the absorption band of 2-G1 centered at 350 nm
and 290 nm decreases. Whereas 2-G1 is stable without
irradiation (Figure S12). The HPLC and HRMS confirmed
the generation of 2-G2 and 2-G4 (Figure S13-14).
ortho-nitrobenzyl derivatives (Scheme 1).41-45 Furthermore,
we prepared several photocages from glycine derivatives
and Sanger’s reagent, and probed their use for
photoinduced release of Zn2+, intracellular imaging of Zn2+
to interrogate the relevant signaling pathways, light
induced regulation of enzymatic activity of Zn2+ depleted
carbonic anhydrase (CA), Zn2+ inhibition of N-methyl-D-
aspartate receptors (NMDARs) in human embryonic kidney
293 (HEK 293) cells, and modulation of the pulse rate of
cardiomyocytes in response to the inhibitory effect of
released tacrine. This investigation led to the discovery of a
new class of photolabile substances derived from Sanger’s
reagent that serve as powerful tools to elicit cellular
functions and signaling pathways.
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
A consideration of the nature of the products enabled us
to propose that the mechanistic route displayed in Figure 1a
is followed in this photochemical process. Specifically, the
excited state of 2 formed by irradiation by UV light
undergoes dehydrative cyclization to produce the acyl-
benzimidazole N-oxide 2-G1, which undergoes secondary
photocleavage to form the simple benzimidazole N-oxide 2-
G2 and eventually amine 2-G4 (through isocyanate 2-G3).
The simulation calculations based on Density Functional
Theory (DFT) and Time Dependent DFT (TD-DFT) were
adopted to support the proposed route of photoreaction.
The results suggest that 2-G1 undergoes the hydrogen
transition from N-H to C of benzimidazole following with
the cleavage of C(benzimidazole)-C(carboxyl) bond,
generating 2-G2 and 2-G3 (Figure S15-17), and 2-G3
readily hydrolyzes to form 2-G4. All of the above
investigations supported that 2-G1 undergoes a secondary
photolysis to produce 2-G2 and 2-G4.
Scheme 1. Synthesis and photoreaction of N-2,4-
dinitrophenylglycine (1).
RESULTS AND DISCUSSION
Mechanism
of
PhotoCleavage
of
N-2,4-
Dinitrophenylglycine amide Derivative Derived from
Sanger’s Reagent. In an earlier investigation,46 Needle et
al demonstrated that N-2,4-dinitrophenylglycine (1),
generated by reaction of Sanger’s reagent and glycine,
undergoes
a UV irradiation promoted reaction that
produces 2-substituted 6-nitrobenzimidazole 1-oxide
(Scheme 1). Based on this finding, we consider if the
photolysis reaction will induce the subsequent cleavage of
amide bond when the carboxyl group of glycine was linked
with amine-bearing moieties. To assess the validity of this
proposal, we prepared the compound, pyridylmethyl and
quinoline containing N-2,4-dinitrophenylglycine amide
derivative 2 (Figure 1a). By using UV-Vis spectroscopic
monitoring, we observed that UV 365 nm irradiation of a
solution of 2 in HEPES buffer (10 mM, pH=7.4) for a 120 s
period causes the intensity of the absorption maximum at
370 nm to decrease and a new band at 285 nm and a
shoulder at 350 nm to concomitantly form (Figure 1b). The
absorption peak at 370 nm is attributed to the R-band
spectrum of 2,4-dinitrophenyl group because the lone-pair
electron of the nitro jumps to π* orbital (n→π*). The
decrease in absorption at 370 nm indicates that at least one
nitro group has transformed. The weakened R band benefit
results in the blue-shift of UV absorption to 350 nm, along
with the K-band absorption peak emerges at 285 nm.
Preliminary information about the products produced in
this photoreaction was gained using HRMS. The results
showed that during the course of irradiation, peaks ascribed
+
to 2 at m/z = 459.1400 (calcd for C23H19N6O5 [2 + H]+,
Figure 1. (a) The proposed photolysis mechanism of Sanger’s
reagent-derived compounds (2, 3, 4, 5, 6 and 7). (b) The
absorbance spectra of 2 (10 µM) with different irradiation time
(365 nm) in HEPES buffer (10 mM, pH=7.4). (c) HRMS of the
compound 2 after irradiation (365 nm).
459.1417) and m/z = 481.1180 (calcd for C23H18N6O5Na+ [2
+ Na]+, 481.1136) decrease, while new peaks at m/z =
145.0740, 271.0788, 441.1252, and 903.2244 arise (Figures
1c and S1). HPLC separation of the photolysis products
followed by HRMS and NMR analysis demonstrated that
three main photoproducts, including 2-G1 (m/z=
To confirm the potential generality of this novel
photocleavage reaction, we prepared the N-2,4-
441.1252),
2-G2
(m/z=271.0788)
and
2-G4
ACS Paragon Plus Environment

Page 3 of 9
Journal of the American Chemical Society
dinitrophenylglycine amide derivative 3 and 4. Indeed,
sanger reagent derivatives, 2i and 4i, and found that the
absence of amide bond eliminates the photo-reactivity
(Figure S25-28), suggesting the critical role of amide bond in
the photolysis mechanism of the Sanger's reagent derived
photo-liable groups.
1
2
3
4
irradiation of a solution of 3 or 4 in HEPES buffer (10 mM,
pH=7.4) led to similar HRMS observations and
characterization of 3(4)-G1, 3(4)-G2 and 3(4)-G4 as major
photoproducts (Figures 1a, S18-24). We further prepared
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 2. Mechanism of photorelease of Zn2+ from 5-Zn2+. (a) Schematic diagram of the complex 5-Zn2+ and uncaging of 5-Zn2+. (b)
The absorption spectra of 5 (10 µM) in HEPES buffer (10 mM, pH 7.4) following different irradiation (365 nm) times. (c) Fluorescence
spectra of 5 (10 µM, 10 mM HEPES buffer, pH 7.4, λex = 450 nm, slits: 5 nm/5 nm) following different irradiation (365 nm) times. (d)
1H NMR spectra of 5 in CD3CN following addition of different amounts of Zn2+. (e) Fluorescence spectra of 10 µM R-1, a fluorescent
sensor developed in our previous syudy,41 in HEPES buffer (10 mM, pH 7.4) containing 5-Zn2+ (10 µM) following different irradiation
(365 nm) times (Inc = 20 s, λex = 315 nm, slits: 5 nm/5 nm). (f) Relative intensities of fluorescence at 497 nm of HEPES buffer solutions
of R-1 (10 µM), R-1 (10 µM) and Zn2+(10 µM), R-1 (10 µM) and 5-Zn2+(10 µM) in the absence of irradiation. Relative intensities of
fluorescence at 497 nm of HEPES buffer solutions of R-1 (10 µM) and 5-Zn2+(10 µM) following irradiation for different times.
The Sanger’s reagent derived N-2,4-dinitrophenylglycine
amide derivative 5, 6 and 7, which were designed to serve
as photocages for the various biological applications
described below, were also prepared and subjected to
photochemical studies. These substances were also found to
undergo the same type of photocleavage process as do 2 and
3 (Figures 1a, S29-32). Importantly, as is demonstrated by
their modest (0.10 for 2 and 0.17 for 3) and high (0.22, 0.28,
0.24 and 0.30 for 4, 5, 6 and 7, respectively, Figure S33-35)
quantum yields, these substances are efficiently cleaved to
produce benzimidazole and amine fragments. We attribute
the electron donating ability of the conjugated groups
(pyridyl < phenyl < propyl) is favorable for photocleavage
efficiency. As a result, the unique photoreaction mechanism
and high quantum yield, along synthetic simplicity make
these substances novel and potentially powerful tools for
constructing photocages.
decarboxylation of a meta-nitrophenylacetic acid derivative,
have shown the advantages of spatiotemporal
controllability
in
interrogating
Zn2+
signaling
pathways.29,30,34,50 However, photocages equipped with a
triggerable fluorophore to sense the release of caged Zn2+
are seldom reported. In this effort, we synthesized
compounds 2, 3, 5, 6 as photocages for Zn2+. Among the four
compounds, 5 and 6 contain a cleavable naphthalimide
fluorophore acting as a fluorescence indicator to monitor
the process of Zn2+ release (Figure 2a). The Zn2+ binding
constants of the compounds were measured respectively
using 4-(2-pyridylazo) resorcinol (PAR) (Figure S36-S38)50-
51
.
Although compounds 2 and 3 exhibit strong
complexation ability to Zn2+ (Kd=9.3 ± 0.7 nM for 2 and
Kd=25.1 ± 1.3 nM for 3), their ability to release Zn2+ is
relatively weak. Especially for compound 2 that holds a
pyridine group and a quinoline group, which strongly binds
to Zn2+ even after photolysis (Table S1). Compounds 5 and
6 show comparable binding affinity to Zn2+ with that of
compound 2 (Kd=11.3 ± 0.6 nM for 5 and Kd=11.0 ± 0.9 nM
for 6), but much higher uncaging efficiency (Table S1).
Therefore, compounds 5 and 6 are used for photocontrolled
Zn2+ release in the following studies.
N-2,4-dinitrophenylglycine amide Based Photocages
for Transport and Release of Zinc Ions. Having
established that N-2,4-dinitrophenylglycine amide
derivative derived from Sanger’s reagent undergo efficient
cleavage upon UV irradiation, we initiated an exploration of
the uses of these substances as photocages for binding to
and release of Zn2+ in biological systems. Given the
importance of Zn2+ in many biological processes including
We observed that the absorption spectrum of 5
undergoes similar UV irradiation promoted changes as does
that of N-2,4-dinitrophenylglycine amide derivative 2
(Figure 2b). In addition, photoreaction of 5 is also attended
by an increase in the intensity of a fluorescence band at 545
nm, which is a consequence of strong intramolecular charge
transfer in the formed product 5-G4 (Figure 2c). That 5
signal transduction, enzyme and immune functions, and
47-49
pathology,
it is of great practical importance to have
available systems for spatially and temporally governed,
safe and targeted regulation of Zn2+. Recently reported Zn2+
photocages, such as NTAdeCage based on the
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 9
Regulation of Biological Function by Zn2+ Photorelease. As
forms a complex with Zn2+ is demonstrated by using HRMS
and 1HNMR titration experiments. HRMS peaks of the
complex 5-Zn2+ (m/z= 723.1009, [5-H+Zn+DMSO]+; m/z =
362.0504, 1/2[5+Zn+DMSO]2+) are observed upon addition
of Zn2+ to a solution containing 5 (Figure S39). Also, the
chemical shifts of the pyridine ring hydrogens in the 1H
NMR spectrum of 5 move to low field, while that of the
amide N-H hydrogen (H13) shifts to the high field upon
addition of Zn2+ (Figure 2d). These changes are caused by
the strong electron-withdrawing effect of Zn2+ and its mode
of complexation involving interactions with the pyridine
group and amide oxygen of 5 (Figure 2a).52
The earlier studied Zn2+-selective fluorescent probe R-153
was used to further evidence photo-triggered Zn2+ release
from 5-Zn2+. It should be noted that excitation at 315 nm
selectively promote fluorescence of R-1 and not of 5-G4
(Figure S40-S41). Addition of 1 equiv. of the 5-Zn2+ to a
HEPES buffer (10 mM, pH 7.4) solution containing R-1 (10
μM) causes an immediate increase the intensity of
fluorescence of R-1 at 497 nm (λex = 315 nm) (Figure 2e),
which is a result of competitive complexation of Zn2+ by R-1
and 5. The intensity of fluorescence at 497 nm increases
upon 365 nm irradiation of the solution, showing that
complexation between R-1 and released Zn2+ takes place
(Figure 2e). After 120 s of irradiation, the emission intensity
at 497 nm becomes close to that associated with complete
formation of R-1-Zn2+ (Figure 2f), exhibiting the high
photoinduced uncaging efficiency of compound 5-Zn2+.
one prototype of applications of Zn2+ photorelease to control
biological processes, we selected the enzyme carbonic
anhydrase (CA) and its hydrolytic activity against 4-
nitrophenyl acetate (NPA) (Figure 3a).54 The enzyme activities
assays demonstrated that Zn2+ depleted carbonic anhydrase
(deCA) is weak active to hydrolysis of NPA, and that addition of
Zn2+ restores its catalytic activity (Figure S42). Photo-triggered
release of Zn2+ from 5-Zn2+, schematically shown in Figure 3a,
was found to partially or fully re-activate deCA in an irradiation
time-controlled manner. Inspection of plots of absorbance at
400 nm, associated with formation of 4-nitrophenol, versus
365 nm irradiation time of HEPES buffer (10 mM, pH 7.4)
solutions containing deCA (2 µM), NPA (125 µM) and 5-Zn2+(5
µM) show that the rates of CA catalyzed hydrolysis of NPA
increase as irradiation time increases (Figures 3b). The
increased catalytic activity is associated with the production of
free Zn2+ needed for conversion of deCA to CA. Moreover, a
linear correlation exists between free Zn2+ production
calculated by CA activity assays and the fluorescence
enhancement during the irradiation (Figures 3c and 3d). These
results showed the photoinduced Zn2+ release can be tuned and
qualified precisely by fluorescent method, and consequently,
allowing the regulation of enzymatic acitivity.
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 4. (a) Schematic for regulation of functions of NMDARs
by irradiation promoted release of zinc ions from 5-Zn2+. (b)
Negative regulation of NMDARs sensitivity by adding
treatment with different agents or combinations of agents. HEK
293T cells were clamped at -70 mV, and 1 mM glutamate and
10 µM glycine were used for all of the whole-cell recordings.
The black line is a negative control that does not contain any
added agents (A two-way ANOVA: no significant influence of
blue line, green line, p > 0.054, a significant effect of purple line,
red line, P<0.001, in 150 s). The green line (a negative control):
with added 100 nM 5-Zn2+ (A two-way ANOVA: no significant
influence of blue line, p > 0.054, a significant effect of purple
line, P<0.01, a significant effect of red line, P<0.001, in 150
seconds). The blue line (a negative control): with added UV-
irradiation (A two-way ANOVA: a significant effect of purple
line, P<0.01, a significant effect of red line, P<0.001, in 150
seconds). The purple line (a positive control): with added 100
nM Zn2+(A two-way ANOVA: no significant influence of red line,
p > 0.054). The red line: with added 5-Zn2+ and UV-irradiation.
For blue and red lines, UV (365 nm) irradiation started at 0 s.
(c) All the peaks are normalized to the baseline, and (i) and (ii)
points of the curves of each color correspond to the respective
(i) and (ii) points in the plots in (b).
Figure 3. (a) Schematic for promotion of the activity of
carbonic anhydrase (CA) for catalysis of the hydrolysis of 4-
nitrophenyl acetate (NPA) by release of Zn2+ from 5-Zn2+. (b)
Plots of absorbance at 400 nm, associated with formation of 4-
nitrophenol versus 365 nm irradiation time of HEPES buffer
(10 mM, pH 7.4) solutions containing deCA (2 µM), NPA (125
µM) and 5-Zn2+(5 µM) for various times. (c) Fluorescence
spectra (λex = 450 nm, slits: 5 nm/5 nm) of 365 nm irradiated
solutions of 5-Zn2+ in HEPES buffer (10 mM, pH 7.4) solutions
for different time periods, recorded 1200 s after adding deCA
(2 µM) and NPA (125 µM). (d) Plots of the concentration of
released Zn2+ (blue dot) and the corresponding fluorescence
intensity of 5-G4 at 545 nm (red dot) versus 365 nm irradiation
times. Inset: plot of the concentration of photoreleased Zn2+
versus fluorescence intensity at 545 nm.
ACS Paragon Plus Environment

Page 5 of 9
Journal of the American Chemical Society
Encouraged by the above results, another experiment
Intracellular Photorelease of Zinc Ions. Effective delivery
to specific intracellular compartments of Zn2+ is essential for
interrogating relevant metal ion governed biological functions
and signaling pathways.59 Sanger’s reagent-based Zn2+
photocages are potentially powerful tools to accomplish this
aim. As a proof-of-concept, N-2,4-dinitrophenylglycine amide
derivative 6, which contains a lysosome directing morpholine
group (Figure 1a), was synthesized and utilized as a photocage
for lysosome targeted delivery of Zn2+. In the initial phase of
this effort, R-1 was utilized to evaluate the ability of 6-Zn2+ to
release Zn2+ in HeLa cells. HeLa cells were incubated with 6-
Zn2+ complex and R-1 at 37 °C for 30 min while irradiating with
UV light and monitoring changes in the intensities of
fluorescence emitted in blue and green channels. The data in
Figure 5a show that along with an enhancement in green
fluorescence associated with formation of 6-G4, blue
fluorescence attributed to R-1-Zn2+ also increases, suggesting
that intracellular release of zinc ions from 6-Zn2+ takes place
upon irradiation. Next, HeLa cells were incubated with 6-Zn2+
and the commercially available lysosomal staining reagent
DND99 at 37 °C for 30 min. Inspection of confocal microscope
images (Figure 5b) demonstrates that the green fluorescence
of 6-G4 increases upon prolonged UV irradiation and co-
localizes with the red fluorescence from DND-99 as indicated
by the orange color in the merged channel. Cytotoxicity tests
also showed that 6-Zn2+ are barely toxic to HeLa cells before
and after UV irradiation (Figure S44). These experiments
demonstrate that 5-Zn2+ targets lysosomes and undergoes
photoinduced intracellular release of Zn2+.
designed to demonstrate the use the complex 5-Zn2+ as a
photocage for release of Zn2+ to a biological function focused
control of N-methyl-D-aspartate receptors (NMDARs), which
are among the major ionotropic glutamate receptors that play
a critical role in excitatory synaptic transmission, synaptic
plasticity, and brain development.55 NMDARs are Ca2+
permeable, voltage-dependent receptors that mediate a slow
synaptic current. One important feature of NMDARs is that they
are negatively regulated by a number of endogenous
extracellular ions including Mg2+, protons and Zn2+.56-58 Thus,
as illustrated in Figure 4a, negative regulation of NMDARs
should be stimulated by irradiation promoted liberation of free
Zn2+ from 5-Zn2+. The sensitivity of a NMDA receptor to
different agents was monitored through whole cell recording
method using HEK 293 cells (Figure 4b and 4c). As shown in
Figure 4b, UV irradiated 5-Zn2+ stimulates formation of a
strong signal, which is comparable to that arising from a
positive control containing added Zn2+. Importantly, NMDARs
only weakly respond to addition of the 5-Zn2+ complex or UV
irradiation alone. While 5-Zn2+ were shown low cytotoxicity
towards HEK 293 cells up to the concentration of 100 μM
(Figure S43). The results clearly demonstrate that photocage 5-
Zn2+ can be employed to effectively stimulate negative
regulation of NMDARs in an irradiation-controlled manner.
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. Tacrine photocontrolled release from compound 7
modulates AChE activity. (a) Schematic diagram of
photocontrolled release of tacrine from compound 7 and its
regulation of AChE activity. (b) Absorption spectra of 7 (10 μM)
in HEPES buffer (10 mM, pH 7.4) irradiated for different times
(365 nm, 0-120 s). (c) Heart rate map of primary rat
cardiomyocytes (see corresponding videos) following
incubation under the following conditions: i-1, ii-1, iii-1 blank
experiments without adding other reagents, i-2, ii-2 and iii-2:
ACh (20 µM); i-3: ACh (20 µM) + ultraviolet light (365 nm) for
60 s; i-4: ACh (20 µM) + ultraviolet light (365 nm) for 60 s +
AChE (0.024 U/mL); ii-3: ACh (20 µM) + 7 (0.2 µM); ii-4: ACh
(20 µM) + 7 (0.2 µM) + AChE (0.024 U/mL); iii-3: ACh (20 µM)
+ 7 (0.2 µM) + ultraviolet light (365 nm) for 60 s; iii-4: ACh (20
µM) + 7 (0.2 µM) + ultraviolet light (365 nm) for 60 s + AChE
(0.024 U/mL); iii-5: ACh (20 µM) + 7 (0.2 µM) + ultraviolet light
(365 nm) for 60 s + AChE (0.072 U/mL, total). Green areas refer
to periods for addition of reagents incubation for 3 min.
Figure 5. Intracellular release of Zn2+ and its fluorescence
imaging. (a) Fluorescence microscope images of HeLa cells
incubated with R-1 (10 μM) and 6-Zn2+ (10 μM) for 30 min.
Bright filed (i); Blue channel (ii); Green channel (iii);
Fluorescence merged (iv). UV irradiation times are 0 s, 10 s, 30
s, 60 s, respectively. (b) Confocal fluorescence images of HeLa
cells incubated with DND99 (10 μM) and 6-Zn2+ (10 μM) for 30
min. Bright filed (i); Red channel (ii); Green channel (iii);
Fluorescence merged (iv). UV irradiation times are 0 s, 30 s, 90
s, respectively. Scale bar = 50 μm.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Extension to Photocontrolled Release of Acetyl exploration of the complex cellular functions and signaling
Page 6 of 9
1
2
3
4
5
6
7
8
Cholinesterase Inhibitor. The photochemical properties
of Sanger’s reagent derived substances can also be utilized
to complex with and release functional species other than
metal ions. Acetylcholinesterase (AchE) and its inhibitor
tacrine were chosen as models to test this suggestion.60 For
this purpose, the Sanger’s reagent derived photolabile N-
2,4-dinitrophenylglycine amide derivative 7 (Figure 1a)
was designed and synthesized as a prodrug to demonstrate
that release of active tacrine can be photopromoted in a
controlled manner, which contains a pyridinic group to
improve the water solubility of compound 7. We observed
that UV irradiation of 7 in HEPES buffer (10 mM, pH 7.4)
produces 7-G4 (tacrine) (Figures 6a and S22) in a process
that is complete within 120 s. The absorption spectra of 7
undergoes similar UV irradiation promoted changes as does
that of N-2,4-dinitrophenylglycine amide derivative 2
(Figure 6b). Next, we showed that released tacrine
pathways.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Chemicals and apparatus; Synthesis routes of com-
pound 1, 2, 3, 4, 2i, 4i, 5, 6 and 7; UV–vis and fluores-
cence assays of the photolysis; HPLC for photolysis
assays; MS spectra of all new compounds and
photoreaction; NMR spectra of all new compounds; Cell
viability assay; Cells experiments of compound5, 6 and 7;
Computational details and Cartesian coordinate.
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
AUTHOR INFORMATION
Corresponding Author
modulates
a biological response in primary mouse
* chenxq@njtech.edu.cn; shiyun@nju.edu.cn
Notes
The authors declare no competing financial interest.
cardiomyocytes containing acetylcholine (ACh) and AChE.
Specifically, upon addition of acetylcholine (ACh), the pulse
rate of mouse myocardial cells drops from 4.4 to 0 beat/s as
a result of binding of ACh to the muscarinic receptor on the
cell membrane61-62 (Figure 6c and Supplementary video i-1,
2). However, addition of AChE to the treated cells leads to
restoration of the initial pulse rate of the cardiomyocytes by
catalyzing the hydrolysis of ACh (Figure 6c and
Supplementary video i-4). Finally, the pulse rate of AChE
and ACh treated cardiomyocytes is increased by adding
additional AChE and 7 followed by UV irradiating for 60 s
(Figure 6c and Supplementary video iii-4, iii-5).
Importantly, neither 7 nor UV irradiation alone have an
effect on the pulse rate (Figure 6c and Supplementary video
i-3, ii-3, iii-3), suggesting that AChE inhibition arises by
photoinduced release of tacrine from 7. Negligible
cytotoxicity of 7, before and after UV irradiation, towards
cardiomyocytes at the testing concentration was observed
(Figure S45).
ACKNOWLEDGMENT
X.C. thanks the support by the National Natural Science
Foundation of China (21722605 and 21978131) and the
National Key R&D Program of China (2018YFA0902200), Y.S.
thanks the support by the National Natural Science Foundation
of China (51776022, 11532015 and 91849112), F. W. thanks
the National Natural Science Foundation of China (21878156),
this work is also supported by the Six Talent Peaks Project in
Jiangsu Province (XCL-034) and the Project of Priority
Academic Program Development of Jiangsu Higher Education
Institutions (PAPD) and the Natural Science Foundation of
Jiangsu Province (BE2019707).
REFERENCES
(1) Sur, S.; Matson, J. B.; Webber, M. J.; Newcomb, C. J.; Stupp, S. I.
Photodynamic Control of Bioactivity in a Nanofiber Matrix. ACS
NANO 2012, 6 (12), 10776–10785.
CONCLUSION
In the investigation described above, we developed new
glycine amide derivatives of Sanger’s reagent as photocages
for Zn2+ and an AChE inhibitor. We demonstrated that the
mechanism for photocleavage of these amides involves
initial cyclization to form acyl-benzoimadazole N-oxides,
which undergo secondary decarboxylation in conjunction
with amide bond cleavage. Moreover, we showed that
amides derived from Sanger’s reagent serve as Zn2+
photocages, which can be employed for light-mediated
control of activity of CA and inhibition of NMDARs. The
ability to apply this strategy more widely was shown by its
use in regulation of the pulse rate of cardiomyocytes by
(2) Haines, L. A.; Karthikan, Rajagopal; Bulent, Ozbas; Daphne A.
Salick; Darrin J. Pochan; Schneider, J. P. Light-Activated Hydrogel
Formation via the Triggered Folding and Self-Assembly of a
Designed Peptide. J. Am. Chem. Soc. 2005, 127, 17025-17029.
(3) Ying, Y. L.; Li, Z. Y.; Hu, Z. L.; Zhang, J.; Meng, F. N.; Cao, C.; Long,
Y. T.; Tian, H. A Time-Resolved Single-Molecular Train Based on
Aerolysin Nanopore. Chem. 2018, 4 (8), 1893-1901.
(4) Bojtar, M.; Kormos, A.; Kis-Petik, K.; Kellermayer, M.; Kele, P.
Green-Light Activatable, Water-Soluble Red-Shifted Coumarin
Photocages. Org. let. 2019, 21 (23), 9410-9414.
(5) Gorka, A. P.; Nani, R. R.; Zhu, J.; Mackem, S.; Schnermann, M. J.
A near-IR uncaging strategy based on cyanine photochemistry. J.
Am. Chem. Soc. 2014, 136 (40), 14153-14159.
using
a Sanger’s reagent derivative that undergoes
photoinduced release of the AChE inhibitor tacrine.
Furthermore, this photocage approach can be employed to
target intracellular compartments and to enable fluorescent
signaling of intracellular processes. The successful proof-of-
concept examples described above open a new avenue for
using Sanger’s reagent-based photolabile compounds in the
(6) Greco, C. T.; Akins, R. E.; Epps, T. H.; Sullivan, M. O. Attenuation
of Maladaptive Responses in Aortic Adventitial Fibroblasts
through Stimuli-Triggered siRNA Release from Lipid Polymer
Nanocomplexes. Advanced biosystems 2017, 1 (8).
(7) Shao, Q.; Xing, B. Photoactive molecules for applications in
molecular imaging and cell biology. Chem. Soc. Rev. 2010, 39 (8),
ACS Paragon Plus Environment

Page 7 of 9
Journal of the American Chemical Society
2835-2846.
Assembly into Nanoparticles. Chem. Sci. 2016, 7 (3), 2392-
2398.
1
2
3
4
5
6
7
8
(8) Chaves, O. A.; Jesus, C. S.; Cruz, P. F.; Sant'Anna, C. M.; Brito, R.
M.; Serpa, C. Evaluation by fluorescence, STD-NMR, docking and
semi-empirical calculations of the o-NBA photo-acid interaction
with BSA. Spectrochimi. acta. A 2016, 169, 175-81.
(25) Roose, M.; Sasaki, I.; Bukhanko, V.; Mallet-Ladeira, S.;
Barba-Barba, R. M.; Ramos-Ortiz, G.; Enriquez-Cabrera, A.;
Farfán, N.; Lacroix, P. G.; Malfant, I. Nitric oxide photo-release
from a ruthenium nitrosyl complex with a 4,4′-bisfluorenyl-
2,2′-bipyridine ligand. Polyhedron 2018, 151, 100-111.
(26) Ellis-Davies, G. C. Caged compounds: photorelease
technology for control of cellular chemistry and physiology.
Nat. methods 2007, 4 (8), 619-628.
(27) Warther, D.; Gug, S.; Specht, A.; Bolze, F.; Nicoud, J. F.;
Mourot, A.; Goeldner, M. Two-photon uncaging: New prospects
in neuroscience and cellular biology. Bioorg. Med. Chem. 2010,
18 (22), 7753-7758.
(9) Jana, A.; Verma, B. K.; Garai, A.; Kondaiah, P.; Chakravarty, A. R.
Mitochondria localizing high-spin iron complexes of curcumin for
photo-induced drug release. Inorg. Chim. Acta 2018, 483, 571-578.
(10) Prusty, D. K.; Adam, V.; Zadegan, R. M.; Irsen, S.; Famulok, M.
Supramolecular aptamer nano-constructs for receptor-mediated
targeting and light-triggered release of chemotherapeutics into
cancer cells. Nat. comm. 2018, 9 (1), 535.
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
(11) Thomsen, H.; Marino, N.; Conoci, S.; Sortino, S.; Ericson, M. B.
Confined photo-release of nitric oxide with simultaneous two-
photon fluorescence tracking in a cellular system. Scientific reports
2018, 8 (1), 9753.
(28) Grenier, V.; Walker, A. S.; Miller, E. W. A Small-Molecule
Photoactivatable Optical Sensor of Transmembrane Potential. J.
Am. Chem. Soc. 2015, 137 (34), 10894-10897.
(12) Wang, H.; Li, W. G.; Zeng, K.; Wu, Y. J.; Zhang, Y.; Xu, T. L.; Chen,
Y. Photocatalysis Enables Visible-Light Uncaging of Bioactive
Molecules in Live Cells. Ange. Chem. Int. Edit. 2019, 58 (2), 561-565.
(29) Bandara, H. M.; Walsh, T. P.; Burdette, S. C. A Second-
generation photocage for Zn2+ inspired by TPEN: characterization
and insight into the uncaging quantum yields of ZinCleav chelators.
Chemistry 2011, 17 (14), 3932-3941.
(13) Park, B. S.; Ryu, H. J. Unique solvent effect on
photochemistry
Tetrahedron Lett. 2010, 51 (11), 1512-1516.
of
ortho-alkylphenacyl
benzoates.
(30) Gwizdala, C.; Singh C. V.; Friss T. R.; MacDonald J. C.; Burdette
S. C. Quantifying factors that influence metal ion release in
photocaged complexes using ZinCast derivatives. Dalton Trans.
2012, 41, 8162–8174.
(14) Kennedy, D. P.; Brown, D. C.; Burdette, S. C. Probing
Nitrobenzhydrol Uncaging Mechanisms Using FerriCast. Org. Lett.
2010, 12 (20), 4486-4489.
(31) Kennedy, D. P.; Gwizdala, C.; Burdette, S. C. Methods for
Preparing Metal Ion Photocages: Application to the Synthesis of
CrownCast. Org. Lett. 2009, 11 (12), 2587-2590.
(15) Hu, Z. L.; Li, Z. Y.; Ying, Y. L.; Zhang, J.; Cao, C.; Long, Y. T.;
Tian, H. Real-Time and Accurate Identification of Single
Oligonucleotide Photoisomers via an Aerolysin Nanopore. Anal.
Chem. 2018, 90 (7), 4268-4272.
(32) Mbatia, H. W.; Bandara, H. M.; Burdette, S. C. CuproCleav-1, a
first generation photocage for Cu+. Chem. Comm. 2012, 48 (43),
5331-3.
(16) Sebej, P.; Wintner, J.; Muller, P.; Slanina, T.; Al Anshori, J.;
Antony, L. A.; Klan, P.; Wirz, J. Fluorescein analogues as
photoremovable
(33) Klan, P.; Solomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.;
Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Photoremovable
protecting groups in chemistry and biology: reaction mechanisms
and efficacy. Chem. Rev. 2013, 113 (1), 119-191.
protecting
groups
absorbing
at
approximately 520 nm. J. Org. Chem. 2013, 78 (5), 1833-1843.
(17) Lu M.; Olesya D. F.; Brent R. M.; Dore, T. M. Bhc-diol as a
Photolabile Protecting Group for Aldehydes and Ketones. Org.
Lett. 2003, 5 (12), 2119-2122.
(34) Basa, P. N.; Barr, C. A.; Oakley, K. M.; Liang, X.; Burdette, S. C.
Zinc Photocages with Improved Photophysical Properties and Cell
Permeability Imparted by Ternary Complex Formation. J. Am.
Chem. Soc. 2019, 141 (30), 12100-12108.
(18) Heather J. M.; Basil P.; Dan F.; Gilles A. L.; Eric J.; Guillemette,
J. G. Photo-Control of Nitric Oxide Synthase Activity Using a Caged
Isoform Specific Inhibitor. Bioorg. Med. Chem. 2002, 10, 1919–
1927.
(19) Takaoka, K.; Tatsu, Y.; Yumoto, N.; Nakajima, T.;
Shimamoto, K. Synthesis and photoreactivity of caged blockers
for glutamate transporters. Bioorg. Med. Chem. Lett. 2003, 13
(5), 965-970.
(20) Takaoka, K.; Tatsu, Y.; Yumoto, N.; Nakajima, T.;
Shimamoto, K. Synthesis of carbamate-type caged derivatives
of a novel glutamate transporter blocker. Bioorg. Med. Chem.
2004, 12 (13), 3687-3694.
(21) Papageorgiou, G.; Ogden, D.; Kelly, G.; Corrie, J. E. Synthetic
and photochemical studies of substituted 1-acyl-7-
nitroindolines. Photoch. Photobio. Sci.2005, 4 (11), 887-896.
(22) Palfi, D.; Chiovini, B.; Szalay, G.; Kaszas, A.; Turi, G. F.;
Katona, G.; Abranyi-Balogh, P.; Szori, M.; Potor, A.; Frigyesi, O.;
Lukacsne H. C.; Szadai, Z.; Madarasz, M.; Vasanits-Zsigrai, A.;
Molnar-Perl, I.; Viskolcz, B.; Csizmadia, I. G.; Mucsi, Z.; Rozsa, B.
High efficiency two-photon uncaging coupled by the correction
of spontaneous hydrolysis. Org. Biomol. Chem. 2018, 16 (11),
1958-1970.
(35) Courts, A. The N-terminal amino acid residues of gelatin. 1.
Intact gelatins. Biochem. J. 1954, 58 (1), 70-74.
(36) Sanger, F. SEQUENCES, SEQUENCES, AND SEQUENCES. Ann.
Rev. Biochem. 1988, 57, 1-28.
(37) Sanger, F.; Thompson, E. O. P. The amino-acid sequence in the
glycyl chain of insulin. II. The investigation of peptides from
enzymic hydrolysates. Biochem. J. 1953, 53 (3), 366-374.
(38) Sanger, F.; Nicklen, S.; Coulson, A. R. DNA sequencing with
chain-terminating inhibitors. Proc. Nati. Acad. Sci. 1977, 74 (12),
5463-5467.
(39) Fu, J.; Wang, Z.; Luo, W.; Xing, S.; Lv, P.; Wang, Z.; Yuan, Z. Novel
Sanger's Reagent-like Styrene Polymer for the Immobilization of
Burkholderia cepacia Lipase. ACS Appl. Mater. Inter. 2018, 10 (37),
30973-30982.
(40) Mottishaw, J. D.; Erck, A. R.; Kramer, J. H.; Sun, H.; Koppang, M.
Electrostatic Potential Maps and Natural Bond Orbital Analysis:
Visualization and Conceptualization of Reactivity in Sanger’s
Reagent. J. Chem. Edu. 2015, 92 (11), 1846-1852.
(41) Horbert, R.; Pinchuk, B.; Davies, P.; Alessi, D.; Peifer, C.
Photoactivatable Prodrugs of Antimelanoma Agent Vemurafenib.
ACS Chem. Biol. 2015, 10 (9), 2099-2107.
(23) Sutton, M. V.; McKinley, M.; Kulasekharan, R.; Popik, V. V.
Photo-cleavable analog of BAPTA for the fast and efficient
release of Ca(2). Chem. Comm. 2017, 53 (41), 5598-5601.
(24) Carling, C. J.; Olejniczak, J.; Foucault-Collet, A.; Collet, G.;
Viger, M. L.; Huu, V. A.; Duggan, B. M.; Almutairi, A. Efficient Red
Light Photo-Uncaging of Active Molecules in Water Upon
(42) Kammari, L.; Solomek, T.; Ngoy, B. P.; Heger, D. Klan, P.
Orthogonal Photocleavage of a Monochromophoric Linker. J. Am.
Chem. Soc. 2010, 132, 11431–11433.
(43) Peters, F. B.; Brock, A.; Wang, J.; Schultz, P. G. Photocleavage
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 9
of the polypeptide backbone by 2-nitrophenylalanine. Chem. Biol.
2009, 16 (2), 148-152.
(54) Mehta, R.; Qureshi, M. H.; Purchal, M. K.; Greer, S. M.; Gong, S.;
Ngo, C.; Que, E. L. A new probe for detecting zinc-bound carbonic
anhydrase in cell lysates and cells. Chem. Comm. 2018, 54 (43),
5442-5445.
1
2
3
4
5
6
7
8
(44) Solomek, T.; Wirz, J.; Klan, P. Searching for Improved
Photoreleasing Abilities of Organic Molecules. Acc. Chem. Res.
2015, 48 (12), 3064-3072.
(55) Mikel Lopez de Armentia; Sah, P. Development and Subunit
Composition of Synaptic NMDA Receptors in the Amygdala: NR2B
Synapses in the Adult Central Amygdala. J. Neurosci. 2003, 23 (17),
6876 – 6883
(45) Yang, Y.; Shao, Q.; Deng, R.; Wang, C.; Teng, X.; Cheng, K.;
Cheng, Z.; Huang, L.; Liu, Z.; Liu, X.; Xing, B. In vitro and in vivo
uncaging and bioluminescence imaging by using photocaged
upconversion nanoparticles. Ange. Chem. Int. Edit. 2012, 51 (13),
3125-3129.
(56) Anderson, C. T.; Radford, R. J.; Zastrow, M. L.; Zhang, D. Y.;
Apfel, U. P.; Lippard, S. J.; Tzounopoulos, T. Modulation of
extrasynaptic NMDA receptors by synaptic and tonic zinc. Proc.
Natl. Acad. Sci. U. S. A. 2015, 112 (20), 2705-2714.
9
(46) Neadle, D. J.; Pollitt, R. J. The Photolysis of N-2,4-
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
Dinitrophenylamino-acids
to
give
2-Substituted
6-
Nitrobenzimidazole I -Oxides. J. Chem. SOC. 1967, 18, 1764-1766.
(57) Fernandez-Marmiesse, A.; Kusumoto, H.; Rekarte, S.; Roca, I.;
Zhang, J.; Myers, S. J.; Traynelis, S. F.; Couce, M. L.; Gutierrez-Solana,
L.; Yuan, H. A novel missense mutation in GRIN2A causes a
nonepileptic neurodevelopmental disorder. Movement Disord.
2018, 33 (6), 992-999.
(47) Chen, Y. C.; Bai, Y.; Han, Z.; He, W. J.; Guo, Z. J.
Photoluminescence imaging of Zn2+ in living systems. Chem. Soc.
Rev. 2015, 44 (14), 4517-4546.
(48) Goldberg, J. M.; Wang, F.; Sessler, C. D.; Vogler, N. W.; Zhang,
D. Y.; Loucks, W. H.; Tzounopoulos, T.; Lippard, S. J.
Photoactivatable Sensors for Detecting Mobile Zinc. J. Am. Chem.
Soc. 2018, 140 (6), 2020-2023.
(58) Gao, K.; Tankovic, A.; Zhang, Y.; Kusumoto, H.; Zhang, J.; Chen,
W.; Shaulsky, G. H.; Hu, C.; Traynelis, S. F.; Yuan, H.; Jiang, Y. A de
novo loss-of-function GRIN2A mutation associated with childhood
focal epilepsy and acquired epileptic aphasia. PlOS one 2017, 12
(2).
(49) Li, J. F.; Yin, C. X.; Huo, F. J. Development of fluorescent zinc
chemosensors based on various fluorophores and their
applications in zinc recognition. Dyes Pigment. 2016, 131, 100-133.
(59) Maret, W. Zinc Biochemistry: From a Single Zinc Enzyme to a
Key Element of Life. Adv. Nutr. 2013, 4 (1), 82-91.
(50) Basa, P. N.; Antala, S.; Dempski, R. E.; Burdette, S. C. A Zinc(II)
Photocage Based on a Decarboxylation Metal Ion Release
Mechanism for Investigating Homeostasis and Biological Signaling.
Ange. Chem. Int. Edit. 2015, 54 (44), 13027-13031.
(60) Fan, L. X.; Wang, J. P.; Meng, F. R.; Luo, Y.; Sui, X.; Zhao, B. Q.;
Li, W. H.; Quan, D. Q.; Yang, J.; Wang, Y. G. Delivering the
Acetylcholine Neurotransmitter by Nanodrugs as an Effective
Treatment for Alzheimer's Disease. J. Biomed. Nanotechnol. 2018,
14 (12), 2066-2076.
(51) Bandara, H. M. D.; Kennedy, D. P.; Akin, E.; Incarvito, C. D.;
Burdette, S. C. Photoinduced Release of Zn2+ with ZinCleav-1: a
Nitrobenzyl-Based Caged Complex. Inorg. Chem. 2009, 48 (17),
8445-8455.
(52) Zhaochao Xu; Kyung-Hwa Baek; Ha Na Kim; Jingnan Cui; Qian,
X.; David R. Spring; Injae Shin; Yoon, J. Zn2+-Triggered Amide
Tautomerization Produces a Highly Zn2+-Selective, Cell-Permeable,
and Ratiometric Fluorescent Sensor. J. Am. Chem. Soc. 2010, 132,
601-610.
(53) Ma, Y.; Wang, F.; Kambam, S.; Chen, X. A quinoline-based
fluorescent chemosensor for distinguishing cadmium from zinc
ions using cysteine as an auxiliary reagent. Sensor. Actuat. B-Chem.
2013, 188, 1116-1122.
(61) Rana, O. R.; Schauerte, P.; Kluttig, R.; Schroder, J. W.; Koenen,
R. R.; Weber, C.; Nolte, K. W.; Weis, J.; Hoffmann, R.; Marx, N.; Saygili,
E. Acetylcholine as an age-dependent non-neuronal source in the
heart. Auton. Neurosci-Basic Clin. 2010, 156, 82-89.
(62) Veerman, C. C.; Mengarelli, I.; Koopman, C. D.; Wilders, R.; van
Amersfoorth, S. C.; Bakker, D.; Wolswinkel, R.; Hababa, M.; de Boer,
T. P.; Guan, K.; Milnes, J.; Lodder, E. M.; Bakkers, J.; Verkerk, A. O.;
Bezzina, C. R. Genetic variation in GNB5 causes bradycardia by
augmenting the cholinergic response via increased acetylcholine-
activated potassium current (I-K,I-ACh). Dis. Model. Mech. 2019, 12
(7), 12.
ACS Paragon Plus Environment

Page 9 of 9
Journal of the American Chemical Society
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
82x43mm (300 x 300 DPI)
ACS Paragon Plus Environment