Dynamics of Oxygen-Independent Photocleavage of Blebbistatin as a One-Photon Blue or Two-Photon Near-Infrared Light-Gated Hydroxyl Radical Photocage

Article abstract of DOI:10.1021/jacs.8b10235

Development of versatile, chemically tunable photocages for photoactivated chemotherapy (PACT) represents an excellent opportunity to address the technical drawbacks of conventional photodynamic therapy (PDT) whose oxygen-dependent nature renders it inadequate in certain therapy contexts such as hypoxic tumors. As an alternative to PDT, oxygen free mechanisms to generate cytotoxic reactive oxygen species (ROS) by visible light cleavable photocages are in demand. Here, we report the detailed mechanisms by which the small molecule blebbistatin acts as a one-photon blue light-gated or two-photon near-infrared light-gated photocage to directly release a hydroxyl radical (^?OH) in the absence of oxygen. By using femtosecond transient absorption spectroscopy and chemoselective ROS fluorescent probes, we analyze the dynamics and fate of blebbistatin during photolysis under blue light. Water-dependent photochemistry reveals a critical process of water-assisted protonation and excited state intramolecular proton transfer (ESIPT) that drives the formation of short-lived intermediates, which surprisingly culminates in the release of ^?OH but not superoxide or singlet oxygen from blebbistatin. CASPT2//CASSCF calculations confirm that hydrogen bonding between water and blebbistatin underpins this process. We further determine that blue light enables blebbistatin to induce mitochondria-dependent apoptosis, an attribute conducive to PACT development. Our work demonstrates blebbistatin as a controllable photocage for ^?OH generation and provides insight into the potential development of novel PACT agents.

Full text of DOI:10.1021/jacs.8b10235

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Article
Dynamics of oxygen-independent photocleavage
of blebbistatin as a one-photon blue or two-photon
near infrared light-gated hydroxyl radical photocage
Ming-De Li, Nai-Kei Wong, Jia Xiao, Ruixue Zhu, Liangliang Wu, Shengyao Dai,
Feng Chen, Guanheng Huang, Xiaoyu Bai, Margarita R Geraskina, Arthur H.
Winter, Xuebo Chen, Yingxia Liu, Wei-hai Fang, Dan Yang, and David Lee Phillips
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10235 • Publication Date (Web): 30 Sep 2018
Downloaded from http://pubs.acs.org on September 30, 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 13
Journal of the American Chemical Society
Dynamics of oxygen-independent photocleavage of blebbistatin as a
one-photon blue or two-photon near infrared light-gated hydroxyl
radical photocage
1
2
3
4
5
6
Ming-De Li,1‡ Nai-Kei Wong, 2,3‡ Jia Xiao,3 Ruixue Zhu,2 Liangliang Wu,4 Shengyao Dai,2 Feng Chen,3
7
8
9
Guanheng Huang,1‡ Xiaoyu Bai,2 Margarita R. Geraskina,5 Arthur H. Winter,5 Xuebo Chen,4* Yingxia
Liu,3 Weihai Fang,4 Dan Yang,2* David Lee Phillips2*
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
1Department of Chemistry and Key Laboratory for Preparation and Application of Ordered Structural
Materials of Guangdong Province, Shantou University, Shantou 515063, China.
2Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
3State Key Discipline of Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen 518112,
China.
4Department of Chemistry, Beijing Normal University, Xin-wai-da-jie 19, Beijing 100875, China.
5Department of Chemistry, Iowa State University, 2101d Hach Hall, Ames, Iowa 50011, United States.
‡These two authors contributed equally to this work.
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
TITLE RUNNING HEAD: Blue light or infrared light triggers hydroxyl radical release from blebbistatin
KEYWORDS Photocage, Photoactivated chemotherapy, two-photo absorption, reactive oxygen species,
blebbistatin, excited state intramolecular proton transfer
Supporting Information Placeholder
ABSTRACT: Development of versatile, chemically tunable photocages for photoactivated chemotherapy (PACT) represents an
excellent opportunity to address the technical drawbacks of conventional photodynamic therapy (PDT) whose oxygen-dependent
nature renders it inadequate in certain therapy contexts such as hypoxic tumors. As an alternative to PDT, oxygen free mechanisms
to generate cytotoxic reactive oxygen species (ROS) by visible light cleavable photocages are in demand. Here, we report the detailed
mechanisms by which the small molecule blebbistatin acts as a one-photon blue light-gated or two-photon near infrared light-gated
photocage to directly release a hydroxyl radical (•OH) in the absence of oxygen. By using femtosecond transient absorption
spectroscopy and chemoselective ROS fluorescent probes, we analyze the dynamics and fate of blebbistatin during photolysis under
blue light. Water-dependent photochemistry reveals a critical process of water-assisted protonation and excited state intramolecular
•
proton transfer (ESIPT) that drives the formation of short-lived intermediates, which surprisingly culminates in the release of OH
but not superoxide or singlet oxygen from blebbistatin. CASPT2//CASSCF calculations confirm that hydrogen bonding between
water and blebbistatin underpins this process. We further determine that blue light enables blebbistatin to induce mitochondria-
dependent apoptosis, an attribute conducive to PACT development. Our work demonstrates blebbistatin as a controllable photocage
for •OH generation and provides insight into the potential development of novel PACT agents.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 13
critical intermediate(s) or photoproducts formed by
blebbistatin under UV or blue light irradiation.19-24
1
INTRODUCTION
Given the intrinsic properties of blebbistatin, we envision
that the presence of a hydroxyl group and carbonyl group
probably allows blebbistatin to undergo excited state
intramolecular proton transfer (ESIPT) with the assistance of
a water bridge since water and hydrogen bonds are ubiquitous
in chemistry and biology. Photoinduced ESIPT is a well-
known photochemical process, which often occurs across a
hydrogen bond.25,26 ESIPT processes produce a tautomer with
an electronic structure distinct from the original excited state,
allowing larger Stokes shifts.27 In this work, a combined
approach of femtosecond transient absorption (fs-TA)
spectroscopy, computational analysis on potential energy
profiles of the excited state, computational prediction of the
absorption spectra of candidate intermediates, and
2
3
4
5
6
7
8
9
Photosensitizers have been a subject of intense study since
the advent of photodynamic therapy (PDT) whose efficacy
has been proven in multiple medical conditions, including
viral infections and malignancies.1-3 The phototoxicity of PDT
agents relies on catalytic conversion of molecular oxygen into
primary reactive oxygen species (ROS), particularly
superoxide or singlet oxygen.4 Of particular import, hydroxyl
radical (•OH) is a common executor of cytotoxicity or
antimicrobial activity of photosensitizers in oxygen-
dependent photochemistry. In solid tumors or under hypoxic
conditions, however, low levels of oxygenation present a
fundamental challenge to PDT applications due to the intrinsic
requirement of dioxygen for ROS generation. In addition,
PDT itself is known to induce acute hypoxia due to the
depletion of oxygen in local tissues, blunting PDT efficacy.5
Thus, it is imperative to develop an oxygen-free strategy to
generate ROS by photoswitchable agents for eliminating
microbial pathogens or cancer cells under hypoxia. In contrast
to PDT, photoactivated chemotherapy (PACT) utilizes
prodrugs shielded by a photocleavable protecting group,
which become bioactive following light-triggered
deprotection. In recent years, the development of novel PACT
agents have focused on d-block metal-based complexes
capable of releasing cytotoxic species against cancer cells.6
While many PACT agents are commonly photoexcitable
under UV to generate ROS, photoswitching at visible light is
desirable for therapeutic development.7-10
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
photoproduct studies by resonance Raman, UV/Vis and H
NMR spectroscopy was employed to verify the mechanisms
underlying blebbistatin photoinactivation. Fluorometric
experiments with several chemoselective ROS sensors further
revealed that hydroxyl radical (•OH) appeared to be noticeably
generated during photolysis of blebbistatin by blue light. •OH
is highly active in inorganic synthesis,28 polymerization,29 and
biomacromolecule modifications.30 Generation of •OH in
solution can be achieved by means of electron pulse radiolysis,
Fenton reaction, tetrachloro-1,4-benzoquinone (TCBQ)
degradation and high-voltage electrical discharge.30-32
Conceptually, the unprecedented one-photon blue light-
triggered or two-photon near infrared-triggered release of •OH
from blebbistatin observed in this study can have several
advantages over traditional methods. Especially, the two-
photon absorption of blebbistatin in the near infrared range
makes it as an excellent potential anti-cancer agent. It also
provides a useful template for the rational design of future
PACT agents and chemical tools such as photocages for
controlled delivery of •OH in living cells and tissues within an
optically attractive therapeutic window. In cell experiments,
blue light irradiation rapidly depolarized blebbistatin-treated
cells, altered mitochondrial morphology, elevated
mitochondrial superoxide production and activated caspase-
related apoptotic pathways. Photoinactivation of blebbistatin
therefore enabled an underappreciated oxidative mode of
cytotoxic action, possibly contingent on the generation of •OH
under blue light. Collectively, we envision that blebbistatin
can be further explored as a potential PACT agent with blue
light and/or intense near infrared light for treating
pathological conditions including skin infections and cancer,
and its photochemical mechanisms revealed herein can
provide valuable insight for the design and development of
future photocaged compounds applicable to the precise and
visible light and/or near infrared light-gated release of •OH in
therapeutic contexts.
In designing novel PACT agents, organic small molecules
are potentially advantageous in that they are highly amenable
to chemical modifications to afford useful attributes such as
aqueous solubility, subcellular targeting, and longer
wavelengths for photoexcitation. Here, we report that
blebbistatin is a blue light or two-photon near infrared light-
cleavable photocage and releases •OH in an oxygen-
independent manner through
mechanism.
a unique photochemical
()-Blebbistatin, a small molecule first identified as a
highly selective, potent reversible non-muscle myosin II
ATPase inhibitor, has been recognized for its utility in
studying the temporal and spatial control of cytokinesis.11-14
Since its discovery, blebbistatin has had a notable impact on
research in biomedicine by virtue of its extensive utilization
in the mechanistic scrutiny of diverse phenomena including
cell migration,11,13,15 axon guidance,16 stem cell self-renewal17
and cell cycle control.18 Intriguingly, blebbistatin becomes
rapidly inactivated upon light irradiation and exhibits
phototoxicity in living cells.19-21 It was speculated that such
photoinactivation (λex=450-490 nm) and phototoxicity of
blebbistatin arose from induction of ROS whose identity
remain unsubstantiated. Mikulich et al. confirmed that UV or
blue light (390-470 nm, peak at 420 nm) was sufficient for
rapidly sensitizing cancer cells to cell death.21 They further
posited that certain ROS other than singlet oxygen were
plausibly involved. Intuitively, the lack of toxicity from
photoreaction products favors the conjecture that irradiation
produces a destructive transient by-product or intermediate
species, or a very reactive moiety on blebbistatin itself.
Although a variety of investigations have attempted to
demystify the phototoxic effects from blebbistatin
Experimental and Computational Methods
Sample preparation. ()-Blebbistatin, dihydrorhodamine
123, hydroxyphenyl fluorescein solution (5 mM) in DMF and
xanthine (X) / xanthine oxidase (XO) were acquired from
commercial sources (Sigma-Aldrich) and were utilized
without any additional treatment. The procedure of synthesis
of HKSOX-1 is as described previously.33 The procedure of
synthesis of HKOH-1 is as described previously.34 Thiourea,
Fe2Cl, H2O2, spectroscopic grade acetonitrile (MeCN), DMF,
potassium phosphate buffer (0.1 M, pH 7.4), D2O and
deionized water were utilized to make up the samples
photoinactivation, there is
a
dearth of mechanistic
understanding about the identity and characteristics of the
2
ACS Paragon Plus Environment

Page 3 of 13
Journal of the American Chemical Society
blebbistatin·5H2O·H3O+ complex in which the hydrogen
employed in the time-resolved spectroscopic measurements.
1
2
3
4
5
6
7
8
bonds could maintain in the range of 1.50~1.80 Å. The same
computational strategy has been repeatedly verified, which
successfully describes the ESIPT reaction over a long distance
in our previous studies.37,38 Ab initio computations were done
employing the complete active space self-consistent field
(CASSCF) method with a 6-31G* basis set and that made use
of a total of 12 electrons in 11 active space orbitals (12e/11o).
The higher energy occupied π, lower energy π* orbitals, the n
orbitals and the related σ/σ* orbitals are contained in the
active space. All of the critical points in the reaction pathways
were obtained by CASSCF optimizations in which a state-
averaged approach with two-roots equally weighted (0.5:0.5)
was used for the singlet excited states, while the triplet and
ground states made use of a single root optimization.
Consideration of the effects of dynamic electron correlation
was done by finding the single-point energy of the optimized
geometries that were recalculated utilizing the second-order
perturbation method (CASPT2) with a five-root state-
averaged CASSCF zeroth-order wave function. All of the
preceding computations were done making use of Gaussian39
and Molcas program packages.40
A. Femtosecond transient absorption (fs-TA)
experiments. The fs-TA measurements were performed with
an apparatus and methods detailed previously35,36 and details
are given in references 45 and 46. Very briefly, fs-TA spectra
were obtained by employing the harmonic (400 nm light) for
the pump pulse and white-light continuum (350800 nm light)
for the probe pulse generated from the 800 nm output of a
femtosecond regenerative amplified Ti:sapphire laser system
For the measurements here, a solution of 40 mL was passed
through a 2 mm thick cell, excited by a 400 nm pump pulse
and then interrogated by the white light continuum pulse.
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
B. Resonance Raman (RR) experiments. The RR
experiments were done by employing a Nd:YAG laser which
supplied the light source at 266 nm that excited a moving
sample of ∼5 × 10−4 M concentration. The RR light was
acquired and detected by a charge-coupled device (CCD). The
RR spectra given here were determined via subtraction of a
solvent without sample spectrum from a corresponding RR
spectrum of solvent with sample primarily to remove solvent
bands.35
C. Fluorometric analysis of ROS. All fluorescence
experiments were acquired at ambient temperature with a
Hitachi F-7000 fluorescence apparatus. The DHR 123,
HKSOX-1, HPF and HKOH-1 probe testing solutions were
photoexcited at 500, 509, 490 and 500 nm, respectively. The
probes were dissolved in DMF to make a 10 mM stock
solution that solvent was added to obtain the required
concentration of testing solutions for measurement. For DHR
E. Cell culture and cytotoxicity assay. Mouse B-16
melanoma cells and human A-375 melanoma cells were
acquired from the Cell Bank of Type Culture Collection of
Chinese Academy of Sciences, Shanghai, China and kept in
RPMI-1640 medium (Gibco) supplemented with 10% FBS
and 1% penicillin/streptomycin for mouse B-16 melanoma
cells and DMEM (high glucose; Gibco) supplemented with 10%
FBS and 1% penicillin/streptomycin for human A-375
melanoma cells. For assessing cytotoxicity of blue light
illuminated blebbistatin, cells were allowed to grow to 70-80%
confluence, and plated into 96-well microplates (Corning) at
a density of 8×104 cells/mL, followed by recovery at 37°C and
5% CO2 for 1618 h. Blebbistatin was dissolved in DMF then
added to a culture medium to obtain a range of concentrations
(0200 µM), in replicates of 5. Typically, the cells were then
placed under a blue LED lamp (λex = 410420 nm; 30 W, ca.
23.8 mW/cm2) with a distance of 10 cm then irradiated 30 min.
In a control plate added with the same range of blebbistatin
doses, the cells were left to stand for 30 min without light
exposure. In experiments involving •OH scavenging, thiourea
of various concentrations (050 mM) was put on the cells
prior to blue light illumination and then the cells were
incubated for another 24 h. At completion of the experiment,
20 µL MTT solution (5 mg/mL) was put into each well and
subsequently incubated for 4 h. The cells were then washed
and incubated with 150 µL DMSO per well. The samples were
gently rocked on an orbital shaker (10-15 min) and then the
450 nm absorbance was measured. Data were analyzed with
Microsoft Excel and GraphPad Prism.
123, HKSOX-1, HPF probe, a LED lamp (30 W) with λex
=
410420 nm was employed to photoexcite the blebbistatin
solutions. The fluorescence intensities of the testing solutions
were recorded after 30 min. For HKOH-1 probe, a LED lamp
(30 W) with λex = 480 nm was employed to photoexcite the
blebbistatin solutions.
D. Two-photon absorption cross-section measurements.
Nonlinear optical properties were characterized by an
open/close-aperture femtosecond Z-scan technique. The
excitation source is
a
Ti:sapphire oscillator-seeded
regenerative amplifier (Coherent, Astrella-Tunable-F-1k),
which gives an output with a pulse energy of 7 mJ at 800 nm
and a repetition rate of 1 kHz. The excitation intensity was
adjusted by a set of natural filters with different transmittances.
The laser beam was focused onto the sample with a beam
waist of ~15 μm. Solution samples with known concentration
were prepared in a quartz cuvette with 1 mm optical path
length. The transmittance of the sample was measured as a
function of input intensity, which was varied by moving the
samples in and out of beam focus along the z-axis. The
aperture before the detector was adjusted to be fully open for
the open aperture Z-scan measurement and 30
%
F. Live cell confocal fluorescence microscopy. The
commercial fluorescent dyes/probes MitoSOX, JC-1,
MitoTracker Deep Red used in this study were obtained from
Life Technologies, and propidium iodide (PI) was from Sigma.
The green-emission superoxide probe HKSOX-133 was a gift
from Prof. Dan Yang. Melanoma cells (5×104 cells/mL) were
plated into 3.5-cm confocal dishes one day before imaging
experiments. Prior to blue light illumination, the cells were
loaded with fluorescent probes for mitochondrial superoxide
MitoSOX (2 µM) or mitochondrial membrane potential probe
JC-1 (2.5 µM), or the organelle-specific dye for mitochondria
MitoTracker Deep Red (20 nM) for 30 min. Then the cells
transmittance of the initial beam for the closed aperture Z-
scan measurement. At least 3 cycles were repeated to
minimize experimental errors and the average values were
calculated to plot out the Z-scan curves.
D. CASPT2//CASSCF calculations. To simulate the
water-assisted ESIPT reaction of blebbistatin, a complex with
five water molecules and
a
hydronium ion, i.e.,
blebbistatin·5H2O·H3O+ was employed to account for the
proton transfers from the donors (O5 and W6) and acceptors
(O3 and N1). Many test calculations were done by utilizing
B3LYP/6-31G* to
find the
structure
of
the
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 13
were washed with HBSS to terminate staining, and were then plated in T25 flasks before treatment. Following overnight
1
2
3
4
5
6
7
8
challenged with blebbistatin in the presence or absence of blue
light (30 min or otherwise specified) in culture medium. PI (1
µg/mL) was added immediately before blue light illumination
of blebbistatin. Images were obtained with a confocal
fluorescence microscope (Zeiss LSM 700). Fluorescence
quantification was done with ImageJ.
recovery, the cells were given blebbistatin (25 µM) with or
without blue light for 30 min. The cells were washed in PBS,
detached by trypsinization and harvested by centrifugation
(600× g, at 4°C for 5 min). To about 2×106 cells of each group,
100 µL of RIPA lysis buffer was added to resuspend and lyse
the cells on ice (15 min). The samples was subjected to
centrifugation at 12000 rpm (Eppendorf Centrifuge 5424 R),
4°C for 15 min. The supernatants were acquired then assayed
for the concentration of protein by BCA method. Caspase 3
G. Flow cytometry (FACS) analysis of cell death.
Melanoma cells were allowed to grow to 70-80% confluence
and then plated into 6-well plates (Corning) at a density of
5×104 cells/mL. After overnight recovery, the cells were
subjected to blebbistatin (25 µM) with or without blue light
present for 30 min. After blue light illumination, the cells
underwent an additional 4 h of incubation. The cells were then
digested briefly with trypsin, followed by neutralization by
fresh serum (30 s). On determining cell density, cells
underwent centrifugation (1200 rpm for 5 min). The
supernatant was disposed of and then about 0.5-1×105 cells
were pelleted in a 5-mL falcon tube. The cells were
resuspended by adding 195 µL of Annexin V-FITC binding
buffer. To each sample, 3 µL of the Annexin V-FITC staining
solution (Beyotime Biotechnology) was added, followed by
gentle rocking. The cells were shielded from light then
underwent incubation on ice for 10 min, and resuspended a
few times to improve staining. Before loading to assess cell
death on a flow cytometer (BD FACSCanto II), the cells were
stained very briefly with PI (1 µg/mL; Sigma). For each
sample, at least 104 cell events were included for analysis.
FACS data were analyzed with FlowJo software.
9
and caspase
8 activities were determined by using
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
colorimetric caspase activity assay kits (Beyotime
Biotechnology) for these enzymes with the help of a standard
curve for the caspase cleavage product pNA (p-nitroaniline).
RESULTS AND DISCUSSION
1. Femtosecond transient absorption studies on the early
events of photophysics and photochemistry of blebbistatin
So as to elucidate the early events of blebbistatin
photoinactivation, ultrafast laser flash photolysis was
employed to record the transient absorption of the excited
states and intermediates of blebbistatin in a 1:1 MeCN:H2O
solution (Figure 1). Several processes were at work after
initial excitation up to 400 ps. During the first several hundred
femtoseconds, the initial excitation of the molecule gave rise
to a singlet excited state, which then quickly evolved into the
S1 excited state with a prominent band around 475 nm. From
1.1 ps to 1.3 ps, the growth of an intermediate with a
noticeable band at 650 nm was observed along with
intensification of a conspicuous absorption at 475 nm (Figure
1a). This can be attributed to a very fast protonation of the
H. Caspase activity assays. Melanoma cells were allowed
to groaw to 70-80% confluence in T75 flasks (Corning) and re-
c
b
0.05
475
475
6.5 ps
25 ps
40 ps
60 ps
80 ps
100 ps
140 ps
220 ps
400 ps
1.3 ps
1.5 ps
2.1 ps
5.0 ps
1.1 ps
0.05
0.04
0.03
0.02
0.01
0.00
0.05
0.04
0.03
0.02
0.01
0.00
1.2 ps
1.3 ps
0.04
0.03
0.02
0.01
0.00
550
350 400 450 500 550 600 650
Wavelength (nm)
350 400 450 500 550 600 650
Wavelength (nm)
350 400 450 500 550 600 650
Wavelength (nm)
d
e
f1.0
0.8
1.0
0.8
0.6
0.4
0.2
0.0
MeCN
MeCN:H2O 1:1
MeCN:H2O 1:1
MeCN:D2O 1:1
MeCN:H2O 1:9
6.5 ps
0.6
350
400
450
500
550
600
650
0.4
0.2
0.0
350 400 450 500 550 600 650
Wavelength (nm)
0
100 200 300 400 500 600 700 800
Time delay (ps)
0
100 200 300 400 500 600 700 800
Time delay (ps)
Figure 1 Shown here are the fs-TA spectra of species produced in a MeCN:H2O 1:1 solution acquired after 400 nm excitation of blebbistatin
from 1.1 ps to 1.3 ps (a), from 1.3 ps to 5.0 ps (b), and from 6.5 ps to 400 ps (c). d, Comparison between the transient absorption at 6.5 ps
(orange) and TD-DFT calculated absorption (dark cyan) of intermediate 2 (see Figure 2 for structure). e, Kinetics of the characteristic
absorption band observed at 475 nm in the fs-TA spectra after 400 nm excitation of blebbistatin in MeCN:H2O 1:1 (squares) and MeCN:D2O
1:1 (circles) solutions. f, The kinetics of the characteristic absorption band observed at 475 nm for the fs-TA spectra after 400 nm excitation
of blebbistatin in MeCN (triangles), MeCN:H2O 1:1 (circles) and MeCN:H2O 1:9 (squares) solutions. The solid lines indicate the fittings to
the data by using a singlet exponential function.
4
ACS Paragon Plus Environment

Page 5 of 13
Journal of the American Chemical Society
cytotoxicity, photosensitivity and fluorescence of blebbistatin
nitrogen atom to produce intermediate 1 (Figure 2a).
1
2
3
4
5
6
7
8
Intermediate 1 then underwent an ESIPT with the aid of water
bridges to produce intermediate 2 (Figure 2a) that had a
characteristic band at 550 nm along with the intense
absorption at 475 nm (Figure 1b). Figure 1d shows that the
experimental transient absorption bands at 6.5 ps agree nicely
with the TD-DFT calculated spectrum of the absorption of the
singlet excited state of intermediate 2. Finally, the transient
having a maximum absorption at 475 nm and weak nearby
absorption at 550 nm evolved into a short-lived epoxide-like
transient 3 (Figure 2a) in which the C=O double bond
chromophore disappeared such that the epoxide-like transient
absorption bands changed to the UV region (calculations
predicted at 237 nm and 316 nm) beyond the detection range
of our fs-TA spectrometer (350-800 nm). Electron transfer
leads to ring-opening of the epoxide-like transient and
formation of intermediate 4, which subsequently underwent
electron transfer to generate the final product with the
concomitant release of •OH (Figure 2a). Because the
photolysis of blebbistatin has been conducted in aqueous
solutions, the source of the electron may mainly come from
the solvent water molecules. That is to say, the water
molecules may be oxidized to serve an electron donor. In
addition, we postulated two alternative photochemical
mechanisms. The first involves initial protonation of N atom
and subsequent aromaticity as a driving force for •OH
formation (Figure 1S). The second involves intramolecular
electron transfer taking place first at the excited state and
were ablated without changing its enzyme inhibitory
activity.22 This evidence strongly suggests that the lone-pair
electrons of nitrogen in pyrrolidine are significantly
delocalized in the benzene ring due to the para-nitro group.
Therefore, the back electron transfer channel is expected to be
weak, which hampers the cleavage of CO bond to produce a
very reactive •OH intermediate in the case of para-
nitroblebbistatin. The exploration of the photophysical and
photochemical reaction mechanisms is to be discussed further
in conjunction with results from CASPT2//CASSCF
calculations (described below).
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
2. Formation of •OH and final product during blebbistatin
photoinactivation
In our study, the final photoinactivated product was examined
with Raman and UV-vis absorption and NMR spectroscopies.
The good agreement of our NMR spectrum of the final
product with the literature reported NMR spectrum24 supports
the notion that overall, blebbistatin appears to lose an oxygen
atom to form the final product after blue light excitation
(Figure 2a). This view is also supported by a significant match
between the calculated absorption and Raman spectra of final
product 1 with the experimental UV-vis absorption and
resonance Raman spectra of photoproducts (Figure 2b and 2c).
Figure 1e presents the kinetics of the diagnostic 475 nm
absorption after 400 nm excitation of blebbistatin in
MeCN:H2O 1:1 and MeCN:D2O 1:1 solutions. The decay time
constants were found to be 78 ps and 107 ps in
MeCN:H2O=1:1 and MeCN:D2O=1:1 solutions, respectively.
This isotope-dependent effect demonstrates that water
molecules are involved during an ESIPT process that
protonates the center ring N atom, which leads to the likely
formation of a short-lived epoxide-like intermediate (Figure
2a). We used piperidine, which is highly chemoselective to
epoxides,41 to test this but no addition of amine to an epoxide
compound was observed in water (data not shown). The
results suggest that the epoxide-like intermediate produced
•
subsequent aromaticity as a driving force for OH formation
(Figure 2S). However, the calculated absorption of the key
intermediates involved in these two mechanisms did not
match with the experimental femtosecond transient
absorption. Recently, Malnasi-Csizmadia and coworkers
designed
a blebbistatin derivative with an electron
withdrawing group (nitro) substituted at C15, the photo- and
a
after
water-assisted
ESIPT
during
blebbistatin
photoinactivation must be a very short-lived species.
Therefore, it appears that a process of ultrafast ring opening
of the epoxide-like intermediate is at work to give the
subsequent species 4 (Figure 2a) to account for its not being
trapped by piperidine. To further clarify how water solvent
participates in the ESIPT process, the kinetics of transient
absorption at 475 nm has been examined after 400 nm
excitation of blebbistatin in varying amounts of water aqueous
solutions (Figure 1f). The decay time constants of 475 nm are
164 ps (in MeCN), 78 ps (in MeCN:H2O=1:1) and 50 ps (in
MeCN:H2O=1:9) solutions. This further confirms that water
molecules participate in the ESIPT process and postulated
formation of the epoxide-like intermediate. In addition, the
formation of the final product 1 also appeared to require water
molecules since the UV-vis absorption of blebbistatin did not
change after illumination in MeCN solvent whereas there was
clear conversion to final product 1 under similar illumination
conditions in aqueous solutions (Figure 3S). In other words,
the photochemical reactions of interest seemed to exclusively
take place with the aid of water molecules. Here, an ESIPT
process can possibly be driven by an increase in the
aromaticity of the final product of blebbistatin.42-44
b
c
225
1622
265
335
*
*
250
300
350
400
450
800
1000
1200
1400
1600
1800
Raman shift (cm-1
)
Wavelength (nm)
Figure 2 Proposed photochemical mechanism for blebbistatin
photoinactivation is shown (a). Shown are the absorption
spectrum of the final product of blebbistatin after 416 nm
excitation in a MeCN:H2O 1:1 solution (top) and a TD-DFT/6-
311G(d,p) predicted absorption spectrum of the final product 1
(bottom) (b). The experimental resonance Raman spectrum (top)
of the final product of blebbistatin observed after 416 nm
excitation and a comparison to the calculated normal Raman
spectrum (bottom) of the final product 1 (c).
3. Detection of ROS formation during blebbistatin
photoinactivation by fluorescence measurement
In a previous biological study by Kolega, the photoproducts
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 13
a
d
b
c
750
600
450
300
150
0
750
600
450
300
150
0
1
2
3
4
5
6
7
8
HKSOX-1 + X/XO with hv
HKSOX-1 + Bleb with hv
HPF + Bleb with hv
HPF with hv
HPF + Bleb without hv
HPF + Bleb with hv (purged
Ar for 30 min)
HPF + Bleb with hv
8000
6000
4000
2000
0
9
520 540 560 580 600 620 640
Wavelength (nm)
500 520 540 560 580 600 620 640
500 520 540 560 580 600 620 640
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
Wavelength (nm)
Wavelength (nm)
λex= 500 nm, λem= 525 nm
e
f
750
800
HPF + Bleb with hv
HPF + Fenton's reagent
HPF only
600
450
300
150
0
600
400
200
HKOH-1 (5 μM)
Blebbistatin (μM)
hν (20 min)
Thiourea (2 mM)
Fenton’s
0.0
0.2
0.4
0.6
0.8
1.0
500 520 540 560 580 600 620 640
Wavelength (nm)
Thiourea (mM)
reagent (2 μM)
Figure 3 The fluorescence experiments were measured in the presence or absence of scavengers in potassium phosphate buffer (0.1 M, pH
7.4, 0.1% DMF). a, Superoxide-dependent fluorescence response (λex = 509 nm) of HKSOX-1 (10 µM) following incubation with a xanthine
(X) / xanthine oxidase (XO) donor system and blue light illuminated blebbistatin (10 µM) for 30 min. b, OH dependent fluorescence
•
response (λex = 490 nm) of HPF (10 µM) following incubation with blue light illuminated blebbistatin (10 µM) under normal aerobic
conditions for 30 min. c, HPF (10 µM) fluorescence response following incubation with blue light illuminated blebbistatin (10 µM) for 30
min with or without Ar purging (30 min). d, HPF (10 µM) fluorescence response (λex = 490 nm) following incubation with blue
light illuminated blebbistatin (10 µM) and different concentrations of thiourea (•OH scavenger; 0, 0.1, 0.25, 0.5, 1 mM). e, HPF (10 µM)
fluorescence response (λex = 490 nm) following reaction with blue light illuminated blebbistatin (10 µM) for 30 min or with Fenton’s reagent
(10 µM Fe2+, 100 µM H2O2) for 30 min. f, Hydroxyl radical dependent fluorescence response (λex = 500 nm) of the •OH-selective fluorescent
probe HKOH-1 (5 µM) following incubation with blebbistatin (5, 25 and 50 µM) under specified conditions for 20 min in the presence or
absence of a •OH scavenger (2 mM thiourea) in potassium phosphate buffer (0.1 M, pH 7.4 0.1% DMF). Photoexcitation of blebbistatin was
conducted at 480 nm. HKOH-1 (5 µM) also reacted with Fenton’s reagent (2 µM) as a technical control.
generated by exhaustive 365 nm or 450-490 nm irradiation of
blebbistatin were found to be nontoxic to cells, even for
conditions of 50 µM and exhaustive exposure for 18 h.19
These photoproducts and their reactive intermediates during
photoinactivation of blebbistatin have since remained
unidentified, with the latter being a more probable source of
phototoxicity. According to the current fs-TA and final
product analysis, the short-lived transients or ROS are more
probable intermediates that can induce the observed cell death.
Experiments done under argon-purged conditions reveal that
involvement of singlet oxygen (1O2) can be ruled out because
oxygen had been reduced to an extremely low level in the
purged solution.45,46 Similarly, peroxynitrite can also be
excluded as a possible reactive intermediate as there was no
loss of nitrogen from blebbistatin. The only plausible ROS
that can theoretically be formed are •OH and superoxide upon
photoexcitation of blebbistatin. To validate this, several
fluorescent probes were used to detect candidate ROS.
Dihydrorhodamine 123 (DHR123) is a fluorescent indicator
for hydrogen peroxide, peroxynitrite, superoxide and •OH.47,48
Blue light illuminated blebbistatin solution readily elicited
a strong fluorescence enhancement of the nonselective ROS
probe DHR123 (Figure 4S), while the same reaction
conditions did not trigger fluorescence enhancement of the
superoxide-selective probe HKSOX-1 (Figure 3a).33
Therefore, superoxide seems an unlikely intermediate
generated via blebbistatin photoinactivation. The well-
established •OH indicator hydroxyphenyl fluorescein (HPF)
is nonfluorescent in its pristine form and fluoresce strongly
upon reaction with •OH or peroxynitrite.48-50 Figure 3b shows
that the fluorescence of HPF was greatly enhanced following
incubation with blue light illuminated blebbistatin (30 min).
Fluorescence enhancement of HPF occurred equally
efficiently under Ar purged and non-Ar purged conditions,
suggesting that singlet oxygen as an intermediate involved is
improbable (Figure 3c). To further verify if •OH is the
candidate ROS species responsible for the phototoxicity of
blue light illuminated blebbistain, thiourea was used as a
selective scavenger to quench OH reactions.51,52 Indeed,
•
fluorescence measurement with HPF suggests a negative
linear relationship between fluorescence intensity of HPF (10
µM) and thiourea concentration (0-1 mM) (Figure 3d). Next,
HKOH-1 as a very selective and sensitive fluorescent probe
•
for detection of OH34 was also used to further confirm the
•
generation of OH after irradiation of blebbistatin with blue
light at 480 nm for 20 min. The fluorescence of HKOH-1 was
significantly enhanced upon photoexcitation of blebbistatin,
while this fluorescence enhancement was readily attenuated
in the presence of 2 mM thiourea (Figure 3f). Based on the
above evidence, it appears that •OH but not singlet oxygen or
superoxide is generated during blebbistatin photoexcitation
with blue light. This surprising observation is nonetheless
consistent with our analysis on the final photoproducts that an
oxygen atom was lost from the initial blebbistatin molecule
(Figures 2b and 2c; Figure 5S and Figure 6S). Recently,
Verhasselt and coworkers postulated that the photosensitizing
6
ACS Paragon Plus Environment

Page 7 of 13
Journal of the American Chemical Society
where 1 GM = 1×1050 cm4 s/photon. Figure 4b shows the
properties might be sufficient to generate hydroxyl or peroxyl
1
radicals as water degradation products in an DMSO:water
mixture upon blue light irradiation,53 though this has not been
tested or proven experimentally. Very importantly, DMSO is
considered inappropriate for studying blebbistatin
photoinactivation as it is a well-documented potent scavenger
for •OH.54 The use of DMSO could potentially interfere with
the photochemical reaction rates. Conventional PDT agents
requires molecular oxygen to generate singlet oxygen. This
work shows that blebbistatin photoinactivation occurs without
need for oxygen (Figures 3c); oxygen levels present a
technical challenge for PDT delivery within the hypoxic
tumor microenvironment. This work, on the other hand,
suggests that blebbistatin should work under even hypoxic or
open/closed aperture Z-scan results of blebbistatin measured
by femtosecond laser pulses at 800 nm. The two-photon
absorption cross-section of blebbistatin was measured to be
667 GM. This indicates that blebbistatin intrinsically has an
excellent two-photon absorption property. Overall, our results
suggest that blebbistatin can be exploited as a photocage with
two-photon excitation in therapeutically appealing regions
(especially near infrared) in biological systems.
2
3
4
5
6
7
8
9
a 3.0
0 min
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
2.5
2.0
1.5
1.0
0.5
0.0
60 min
anoxic environments,
a proposition deserving future
validation by biologists in the PACT field. It is noteworthy
•
that the blue light dependent photo-release of OH from
blebbistatin offers significant advantages over •OH generation
•
by UV photolysis or Fenton reaction. OH generation by
photolysis depends on high energy UV light which invariably
elicits photodamage and photodecomposition of biological
molecules in living cells and in vivo samples. Fenton reaction
200 250 300 350 400 450 500 550
Wavelength (nm)
b
•
requires fresh preparations and the production of OH is
1.000
highly precipitous and hard to harness in a controlled manner.
In contrast, photoexcitation of blebbistatin permits control
over the rate and amount of OH release via adjusting the
source of light used (Figure 7S), with blue light being a form
of irradiation therapeutically superior to UV. The
fluorescence enhancement of HPF by photoexcitation of
blebbistatin was also found to be more efficient than that by
Fenton’s reagent when used at a comparable concentration
(Figure 3e).
0.996
0.992
0.988
•
-30 -20 -10
0
10
20
30
40
4. Two-photon excitation (800 nm) of blebbistatin in an
aqueous solution
Z-position (mm)
Figure 4 UV-vis absorption spectra evolution of blebbistatin in
aqueous solution after two-photon irradiation at 800 nm (a) and
the open/close aperture Z-scan results of blebbistatin measured
by femtosecond laser pulses at 800 nm (b).
Although blebbistatin can undergo blue light-triggered
photocleavage to yield its final product in an aqueous solution,
blue light has relatively small penetration depth, which may
restrain blebbistatin’s potential for PACT applications. We
envisage that if the compound can instead be photoexcited by
a two-photon process with wavelengths above 800 nm, it
would be very useful for the development of PACT capable of
reaching tissues of greater depth. Figure 4a shows the UV-vis
absorption evolution of blebbistatin excited by near infrared
light (800 nm) with 180 mW laser power. Evidently,
blebbistatin can efficiently undergo two-photon excitation
triggered photochemical reaction to produce the final product,
as observed with one-photon blue light excitation (Figure 3S
(right)). This strongly suggests that blebbistatin can
photorelease •OH by two-photon excitation, whose excitation
wavelength is in near infrared region, which is fairly efficient
in penetrating living cells and tissues. To explore the two-
photon absorption properties of blebbistatin, the samples were
measured by an open/closed aperture Z-scan technique, with
an 800 nm pulsed laser and a repetition rate of 1 kHz. The
position of the sample cell can be altered along the z-axis (the
laser-beam direction), while obtaining the transmittance
changes in the far field. β is the nonlinear absorption
coefficient. The two-photon absorption coefficient β is
obtained by fitting the experimental transmittance data. The
two-photon absorption cross-section (δ) is thus calculated by
using the equation δ = hωβ/(2πN0), where h휔/2π is the
excitation photon energy and N0 is the number of molecules
per cm3. δ is generally presented in GM (Göpert-Meyer) units,
5. CASPT2//CASSCF calculations for photochemical
mechanisms underlying blebbistatin photoinactivation
Our mass and NMR spectroscopic results (Figure 5S and 6S)
show that the difference of the relative molecular mass is the
weight of one oxygen atom (16) between the initial reactant
and the N1 protonated product. This indicates that N1
protonation assisted by ESIPT reaction is a required step
through the introduction of an additional proton from the
aqueous solvation. Therefore, the nH2O∙H3O+ complex was
employed to serve as the donor of the ESIPT reaction, which
can be used to mimic the water solution surrounding it for the
•
direct release of OH in the presence of an appropriate water
hydrogen bonding network. Computational tests reveal that
the stabilization energy reaches its maximum (ca. 30 kcal/mol)
by the introduction of
6
H2O molecules, i.e.,
blebbistatin∙5(H2O)∙H3O+ and remains almost unchanged with
the participation of more water molecules (see Table 1S).
Consequently, the CASPT2//CASSCF calculations of
blebbistatin∙5(H2O)∙H3O+ complex were performed to
examine the minimum energy profile associated with the
photochemistry of blebbistatin. This serves to further
elucidate how the water-assisted ESIPT process drives
blebbistatin photoinactivation to release •OH and produces the
inactive final product 1. As shown in Figure 5, upon
photoexcitation at 419 nm, blebbistatin is instantaneously
populated in the Frank-Condon region of strongly allowed SCT
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 13
a
1
2
3
b
SCT(1ππ*)
SCT(1ππ*)-FC
S0
TS2
4
5
6
7
8
9
TS1
60
45
30
15
0
CI(SCT/S0)
·OH
SCT(1ππ*)-Min
INT-1

SET
W3
OH−
W2
W4
W1
H7
O3
H6
W5
O5
hν
419nm
C4
C2
W6
INT-2
N1
H8
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
S0
OH anion
8
Diradical
0
2
4
6
10
12
14
Intrinsic Reaction Coordinate / amu1/2 · Bohr
Figure 5 Shown are the minimum energy profiles based on integration of fs-TA spectroscopic data and CASPT2//CASSCF calculations (a).
Proposed mechanistic pathway of blebbistatin•5H2O•H3O+ (b).
(1ππ*) state (f = 0.18) that initiates the charge transfer along a
desired direction from the tolyl ring to the center 3-
hydroxypyridin-4(3H)-one ring. As a result, a noticeable
negative charge significantly accumulates around N1 of the 3-
hydroxypyridin-4(3H)-one ring and carbonyl C2=O3 (refer to
numbered atoms in Figure 5), which facilitates the occurrence
of the subsequent ESIPT reaction aided by water. Consistently,
a tiny barrier (TS1, 3.6 kcal/mol) is found in the N1
protonation pathway of the SCT (1ππ*) state, producing the first
intermediate (INT-1). N1 protonation leads to a slight
decrease in the accumulation of negative charge around the
proton acceptor carbonyl O3 from 0.68 in the minimum of
SCT (1ππ*) state, SCT (1ππ*)-Min, to 0.64 in INT-1.
Accordingly, a moderate barrier (TS2, 9.1 kcal/mol) is
determined in the second step of ESIPT reaction, i.e.,
protonation of carbonyl C2=O3 with the concomitant
deprotonation of hydroxyl O5-H6.
negative charge. This leads to the instant ring opening of the
epoxide while the heterolysis of C2O5 is triggered
simultaneously along a concerted reaction path. In an aqueous
solution in which the surrounding environment of blebbistatin
is water, water plays a pivotal role in opening the epoxide-like
intermediate. This is the main reason why no addition of
amine (piperidine) to an epoxide compound was observed in
aqueous solutions. By surmounting an 18.2 kcal/mol barrier,
an OH anion is finally released with the formation of a cation
in the conjugation ring moiety along the reaction path of
C2O5 bond heterolysis. Meanwhile, the energy level for the
generation of •OH decreases progressively and approaches the
formation path of OH- anion. The energy gap is narrowed
eventually to 5.3 kcal/mol between the two channels. This
•
indicates that the OH is likely produced through single
electron transfer (SET) from the OH anion to the center
pyridine ring by overcoming a tiny barrier. The present
computational results demonstrate that photoexcitation of
As shown in Figure 5, although deprotonation of hydroxyl
O5-H6 is a driving force of the ESIPT reaction, protonation
of the carbonyl group and N1 is done by the water solvent.
This finding is also corroborated by the labeling experiments,
as shown in Figure 1e. With the accomplishment of
deprotonation of hydroxyl O5H6, a negative charge center is
bared around O5 which becomes ready for nucleophilic attack
on carbocation of C2. On the other hand, the released proton
from the hydroxyl O5H6 simultaneously drives a series of
proton translocations toward the oxygen of carbonyl C2=O3
via a water chain, producing the enol form of C2-O3-H. As a
result, the carbocation ion C2+ is generated through an
effective charge translocation from C2 to O5 along a water
•
blebbistatin leads to coexistence of OH anion and OH. No
•
electron paramagnetic resonance (EPR) signal for OH was
detected (Figure 8S), though •OH could nonetheless be
visualized by the chemoselective fluorescent probes HPF and
HKOH-1 (Figure 3b and 3f). Intrinsically, fluorescence
measurement affords superior sensitivity and selectivity,
•
thereby enabling OH detection of extremely low analytic
levels beyond the detection limit of EPR.
6. Potentiation of cytotoxicity of blebbistatin by blue light.
Owing to its unusual photochemical attributes and efficient
photoconversion in the presence of blue light, blebbistatin is
anticipated to operate as an oxidant generator in the cellular
environment. While our chemical validation results do not
support the in vitro formation of superoxide or singlet oxygen
(Figure 3a and 3c), which are otherwise well-documented
ROS formed in photodynamic experiments with UV
irradiation, the observed atypical •OH formation in an aqueous
chemical system implies that blebbistatin could
photochemically behave in similar manners in living cells,
thereby rapidly modifying cellular components through
oxidation. We attempted to validate this effect in skin cancer
cells of two types. In the presence of blue light (refer to
Methods on conditions for cytotoxicity assay), blebbistatin
exhibited increased cytotoxicity toward A375 human
melanoma cells and B16 mouse melanoma cells (Figure 6a).
wire. Obviously, the formed C2+O5
pair is
considerably stabilized by the introduced water wire through
the strong hydrogen bond interactions between the water
molecules and C2-O3-H, the bare O5 and N1H moieties.
Meanwhile, the energetic level of ground state gradually
increases and eventually reaches the conical intersection of CI
(SCT/S0) between SCT (1ππ*) and ground state that functions as
an effective relay to enable a very fast combination of C2+ and
O5 in the ground state, giving rise to the formation of a short-
lived epoxide-like intermediate INT-2, whose energy is 29.4
kcal/mol less than that of the CI (SCT/S0). Once the active INT-
2 is formed, the proton H7 backward transfer is immediately
initiated due to the strong attraction of the O5 that has some
8
ACS Paragon Plus Environment

Page 9 of 13
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
Figure 6 Potentiation of cytotoxicity of blebbistatin by blue light. a, Evaluation of cytotoxicity of blebbistatin in the presence or absence of
blue light (hv) in B16 mouse melanoma cells and A375 human melanoma cells by MTT assay. Results are representative of three independent
experiments. b, Time-lapse confocal imaging of B16 cells after exposure to blebbistatin and blue light (30 min). Cells were stained with
propidium iodide (PI) imaging before imaging. Scale bar = 100 µm. c, Quantitation of PI fluorescence intensities in cells treated with
blebbistatin at 25 µM in b with additional controls. d, Time-lapse confocal imaging of PI-stained B16 cells exposed to varying durations of
treatment with blebbistatin (25 µM) and blue light. Scale bar = 200 µm. e, Quantitation of fluorescence intensities of cells in d. f, Effects of
thiourea on cell death induced by blebbistatin and blue light. Cells were first pre-incubated (30 min) with thiourea at indicated concentrations
and then subjected to challenge of blebbistatin (25 µM) and blue light (30 min). Statistical significance was determined at ***p < 0.001 by
Student’s t test.
In MTT assay, blue light irradiation resulted in a reduction
of >2.6 and >4.1 folds in IC50 values for A375 and B16 cells,
respectively. It was anticipated that blue light illuminated
blebbistatin extends its effects by increasing cell permeability.
Indeed, in time-lapse confocal imaging (Figure 6b), blue light
illuminated blebbistatin (10 and 25 µM) caused a loss in
plasma membrane integrity in B16 cells to a degree that
depends on the dose and time of exposure to light, as assessed
by propidium iodide (PI) staining. At 25 µM, blue light
illuminated blebbistatin was most effective and induced a
maximal increase in cell permeability in B16 cells from 6h
post-treatment (Figure 6c). To evaluate how efficiently blue
light induced a loss of the integrity of the plasma membrane
in blebbistatin-treated cells, B16 cells were exposed to blue
light for various time durations (5, 15 and 30 min). The results
suggest that blue light illuminated blebbistatin approached its
maximal effects by 30 min (Figure 6d). PI staining increased
by about 8 times upon blue light irradiation, relative to
baseline of the untreated control (Figure 6e). We anticipated
that the observed cytotoxicity by blue light illuminated
blebbistatin can be partially reduced by thiourea, an
undergo photochemical reaction in the visible light regions.
Because thiourea (in mM range) reacts with a downstream
photoproduct (•OH ) of the blebbistatin photoinactivation
•
process, it is anticipated to act as a sink for OH (known to
occur in equilibrium in pM range) without effects on the
photochemistry of blebbistatin in the chemical system. Indeed,
in MTT assay, thiourea pretreatment (30 min) significantly
improved the viability of B16 and C375 cells exposed to blue
light and blebbistatin (25 µM, 30 min) (Figure 6f).
7. Flow cytometry analysis of cell death during blue light
illumination of blebbistatin
Identifying the types of cell death induced by blue light
illuminated blebbistatin is of interest, because non-pyrogenic
cell death such as apoptosis generally elicits minimal local
inflammation and therefore less adverse effect in therapeutic
contexts. In order to further clarify the effects of blue light
illuminated blebbistatin (25 µM) at the cellular level, we
performed flow cytometry (FACS) analysis in C375 and B16
cells to elucidate the types of cell, death brought about. As
shown in Figure 7a, 30 min exposure to blue light illuminated
blebbistatin (bleb + hv) resulted in a significant increase in PI-
and Annexin A5 double-positive cells as well as Annexin A5
mono-positive cells by 4 h posttreatment, indicating increased
cell death of late apoptosis and early apoptosis, respectively.
Consistent with this observation, blue light illuminated
blebbistatin (25 µM; 30 min) rapidly induced a disappearance
of mitochondrial membrane activity, an initial part in the
•
established scavenger for OH . It has been established that
thiourea is highly recalcitrant to UV irradiation and does not
undergo any photochemical reactions.55 We also validated the
UV absorption spectra of thiourea in acetonitrile (see Figure
9S), which has an absorption peak squarely in the UV regions
(< 300 nm), thus precluding any of chance of thiourea to
9
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 13
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 7 Analysis of cell death during blue light illumination of blebbistatin. a, A375 and B16 Cells were challenged with blebbistatin (25
µM) alone, blue light alone, or blebbistatin and blue light for 30 min, and briefly stained with PI and Annexin A5 for flow cytometry analysis.
b, Representative confocal images of C375 cells treated with blebbistatin (25 µM) in the presence of absence of blue light (30 min), and
stained with JC-1 (2.5 µM). Scale bar = 50 µm. (Inset) Quantification of red-to-green fluorescence emission ratios of cells (n ≥ 207) in b. c,
Effects of blebbistatin (25 µM) and blue light (30 min) on mitochondrial superoxide formation in C375 cells, as visualized by MitoSOX (2
µM) in confocal imaging. Scale bars = 10 µm. (Inset) Quantification of fluorescence intensities in cells (n ≥ 133) in c. d, Representative
confocal images of mitochondrial morphology of C375 cells stained with MitoTracker Deep Red (20 nM) with (right) or without (left)
treatment of blebbistatin (25 µM) and blue light. Scale bar = 5 µM. e, Response of A375 cells and B16 cells to blue light alone, blebbistatin
alone, or blebbistain (25 µM) and blue light (30 min) in a quantitative assay for caspase-3 activity. f, Response of A375 cells and B16 cells
to blue light alone, blebbistatin alone, or blebbistain (25 µM) and blue light (30 min) in a quantitative assay for caspase-8 activity. Statistical
significance was determined at **p < 0.01 and ***p < 0.001 by Student’s t test.
apoptotic cascade in C375 cells, as assessed by the membrane-
potential sensitive dye JC-1 (2.5 µM) (Figure 7b).
Mitochondrial superoxide formation another indicator for
activation of mitochondrial-dependent apoptosis, was
strongly induced by blue light illuminated blebbistatin (25 µM;
30 min) in C375 cells as measured by the established
mitochondrial superoxide probe MitoSOX (2 µM) (Figure 7c).
In addition, blue light illuminated blebbistatin (25 µM; 30 min)
treatment also resulted in marked changes in mitochondrial
morphology in C375 cells, as monitored with MitoTracker
Deep Red (20 nM). Exposure to blue light and blebbistatin
rapidly increased fissure of mitochondria, which displayed
toroidal (donut-like) features suggestive of a stressed state
instead of tubular morphology characteristic of resting
mitochondria (Figure 7d). Lastly, quantitative caspase activity
assays were performed to determine the apoptotic mediators
involved. The results suggest that in both C375 and B16 cells,
blue light illuminated blebbistatin (25 µM; 30 min)
significantly upregulated the action of caspase-3 (Figure 7e)
and caspase-8 (Figure 7f) by 4 h posttreatment, further
confirming that blebbistatin potently induced apoptosis in
melanoma cells in the presence of blue light. Our results
indicate that blue wavelength photon illumination of
blebbistatin is
a robust approach to rapidly induce
mitochondria-dependent apoptosis in melanoma cells, which
presents features reminiscent of cell death induced by other
PDT agents.56,57 However, the unique photochemical
characteristic of blebbistatin to directly generate •OH is likely
to be important to its enhanced cytotoxic effects, which
distinguishes blebbistatin from conventional PDT agents for
which singlet oxygen or superoxide are the initial or primary
ROS. Based on our experimental results, blebbistatin
therefore operates not as a typical photosensitizer but rather a
photocage or PACT agent. Our cell imaging studies (e.g. loss
of mitochondrial membrane potential, mitochondrial
morphological changes, etc.) suggest that although
10
ACS Paragon Plus Environment

Page 11 of 13
Journal of the American Chemical Society
blebbistatin itself does not release superoxide (Figure 3a),
directly released OH from blebbistatin potentially sets off a
cascade of cell stress events that include the production of
intracellular ROS (e.g. mitochondrial superoxide, Figure 7c),
which could perpetuate the cell damage leading the eventual
cell death observed (Figures 6a and 7a).
Corresponding Authors
•
1
2
3
4
5
6
7
8
E-mail addresses: phillips@hku.hk, yangdan@hku.hk,
xuebochen@bnu.edu.cn
ACKNOWLEDGMENTS
Partial support from the Hong Kong Research Grants Council
grants GRF 17307916, AoE/P-03/08, SEG HKU/07, AoE/P-
705/16 , The University of Hong Kong Development Fund 2013-
2014 project “New Ultrafast Spectroscopy Experiments for
Shared Facilities”, Shantou University Initial Funding
(NTF16010) and Natural Science Foundation of China
(21773151, 81570552, 21725303), The Science and Technology
CONCLUSION
The present study on the photochemical mechanisms
underlying blue light photoinactivation of blebbistatin has
revealed an unexpected mode of blebbistatin phototoxicity, in
which water-assisted ESIPT enables an unprecedented direct
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
•
Innovation
Commission
of
Shenzhen
grant
photo-release of OH from blebbistatin in aqueous solutions.
(JCYJ20160427151920801) and Special Support Funds of
Shenzhen for Introduced High-Level Medical Team grants
(ZDSYS201504301534057,
acknowledged. We thank the useful suggestion from Dr. Jacob
Jun Hu and Dr. Tao Su.
This process bypasses the generation of singlet oxygen or
superoxide as would be anticipated in conventional PDT. Our
results suggest that such photo-release of •OH involves
hydrogen bonding between water molecules and blebbistatin
via concerted protonation and ESIPT, which allows
blebbistatin to structurally evolve along a energetically
favorable pathway on the potential energy landscape to
release •OH. Efficient blue light photoconversion of
blebbistatin suggests that this photo-labile compound can be
exploited as a useful photocage for controlled release of •OH,
SZSM201512005)
are
REFERENCES
(1)Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer
2003, 3, 380-387.
(2) Moore, C. M.; Pendse, D.; Emberton, M. Nat. Clin. Pract. Urol. 2009,
6, 18-30.
(3) W. Piao, K. Hanaoka, T. Fujisawa, S. Takeuchi, T. Komatsu, T. Ueno,
T. Terai, T. Tahara, T. Nagano, Y. Urano. J. Am. Chem. Soc. 2017, 139,
13713−13719
(4) Castano, A. P.; Mroz, P.; Hamblin, M. R. Nat. Rev. Cancer 2006, 6,
535-545.
(5) V. H. S. van Rixel, G. F. Moolenaar, M. A. Siegler, L. Messori, S.
Bonnet. Dalton Trans., 2018, 47, 507-516.
(6) N. J. Farrer, L. Salassa P. J. Sadler. Dalton Trans., 2009, 0, 10690-
10701
(7) Bonnet S. Dalton Trans. 2018, 47, 10330-10343.
(8) Mari C, Pierroz V, Ferrari S, Gasser G. Chem. Sci. 2015, 6, 2660-
2686.
•
with practical advantage over conventional mode of OH
generation such as Fenton reaction. Remarkably, blebbistatin
is efficiently photocleaved by two-photon photoexcitation as
evidenced in absorption spectra which means that near
•
infrared light can also trigger the cage to photorelease OH.
Blebbistatin can therefore act potentially as a useful PACT
agent in therapy development. To the best of our knowledge,
there have been no successful attempts in delivering Fenton
reagents (namely, free iron (II) and H2O2 at 100500 µM) to
target tissue sites as a therapeutic strategy. Most exogenous
iron in biological systems are quickly assimilated by iron
carrier proteins such as ferritin and transferrin as a cellular
protective response. In addition, conventional PDT agents are
functionally dependent on molecular oxygen to generate
singlet oxygen. For drug delivery to deep tissues or hypoxic
tumor microenvironment, this presents technical challenges
and limits the efficacy of conventional PDT agents. On the
other hand, our fs-TA spectroscopic findings on blebbistatin
clearly demonstrate that photorelease of •OH and blebbistatin
photoinactivation occur without the need for molecular
oxygen, which potentially is an advantage distinctive of
blebbistatin photochemistry. Collectively, our results have
revealed an underappreciated mechanism by which small-
molecule compounds can be efficiently transformed in
therapeutically desirable regions of the excitation/emission
spectra. We find it exciting to speculate that blebbistatin’s
photochemical attributes can have practical implications in
PACT for treating refractory conditions such as skin cancers
and microbial infections. As a blue light-gated photocage for
•OH generation, blebbistatin can also serve as a conceptual
blueprint for rational design and development of new PACT
agents. These will be worthwhile areas for future investigation.
(9)Presa A, Brissos RF, Caballero AB, Borilovic I, Korrodi-Gregório L,
Pérez-Tomás R, Roubeau O, Gamez P. Angew. Chem. Int. Ed. 2015, 54,
4561-4565.
(10) Lan, G.; Ni, K.; Xu, Z.; Veroneau, S. S.; Song, Y.; Lin, W. J. Am.
Chem. Soc. 2018, 140, 5670–5673.
(11) Straight, A. F.; Cheung, A.; Limouze, J.; Chen, I.; Westwood, N. J.;
Sellers, J. R.; Mitchison, T. J. Science 2003, 299, 1743-1747.
(12) Allingham, J.; Smith, R.; Rayment, I. Nat. Struct. Mol. Biol. 2005,
12, 378-379.
(13) Paszek, M. J.; Dufort, C. C.; Rossier, O.; Bainer, R.; Mouw J. K.;
Godula, K.; Hudak, J. E.; Lakins, J. N.; Wijekoon A. C.; Cassereau, L.;
Rubashkin, M. G.; Magbanua M. J.; Thorn, K. S.; Davidson, M. W.;
Rugo, H. S.; Park, J. W.; Hammer, D. A.; Giannone, G.; Bertozzi, C.
R.; Weaver, V. M. Nature 2014, 511, 319-337.
(14) Wilson, C. A.; Tsuchida, M. A.; Allen, G. M.; Barnhart, E. L.;
Applegate, K. T.; Yam, P. T.; Ji, L.; Keren, K.; Danuser, G.; Theriot, J.
A. Nature 2010, 465, 373-379.
(15) Kuo, J. C.; Han, X.; Hsiao, C. T.; Yates, J. R.; Waterman, C. M.
Nat.Cell Biol. 2011, 13, 383-393.
(16) Medeiros, N. A.; Burnette, D. T.; Forscher, P. Nat. Cell Biol. 2006, 8,
215-226.
(17) Walker, A.; Su, H.; Conti, M. A.; Harb, N.; Adelstein, R. S.; Sato, N.
Nat. Commun. 2010, 1, 71.
(18) Rosenblatt, J.; Cramer, L. P.; Baum, B.; McGee, K. M. Cell 2004,
117, 361-372.
(19) Kolega, J. Biochem. Biophys. Res. Commun. 2004, 320, 1020-1025.
(20) Sakamoto, T.; Limouze, J.; Combs, C. A.; Straight, A. F.; Sellers, J.
R. Biochemistry 2005, 44, 584-588.
ASSOCIFATED CONTENT
Supporting Information.
“This material is available free of charge via the Internet at
http://pubs.acs.org.”
(21) Mikulich, A.; Kavaliauskiene, S.; Juzenas, P. Biochimica. Biophysica.
Acta. 2012, 1820, 870-877.
(22) Képiró, M.; Várkuti, B. H.; Végner, L.; Vörös, G.; Hegyi, G.; Varga,
M.; Málnási-Csizmadia, A. Angew. Chem. Int. Ed. 2014, 53, 8211-8215.
(23) Lucas-Lopez, C.; Allingham, J. S.; Lebl, T.; Lawson, C. P. A. T.;
Brenk, R.; Sellers, J. R.; Rayment, I.; Westwood, N. J. Org. Biomol.
AUTHOR INFORMATION
11
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 13
Chem. 2008, 6, 2076-2084.
Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
(24) Képiró, M.; Várkuti, B. H.; Bodor, A.; Hegyi, G.; Drahos, L.; Kovács,
M.; Málnási-Csizmadia, A. Proc. Natl. Acad. Sci. 2012, 109, 9402-
9407.
(25) Heberle, J.; Dencher, N. A. Proton Transfer in Hydrogen-Bonded
Systems; Bountis, T., Plenum Press, New York, 1992, Vol. 291, pp
187–197.
(26) Padalkar, V. S.; Seki, S. Chem. Soc. Rev. 2016, 45, 169-202.
(27) Demchenko, A. P.; Tang, K.-C.; Chou, P. T. Chem. Soc. Rev. 2013,
42, 1379.
(28) Zard, S. Z. Radical reactions in organic synthesis; Oxford University
Press, USA, 2003, Vol. 28, pp 364-368.
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari,
K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.;
Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.;
Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.;
Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.
Gaussian, Inc., Wallingford CT, 2004.
(40) Karlström, G.; Lindh, R.; Malmqvist, P. A.; Roos, B. O.; Ryde, U.;
Veryazov, V.; Widmark, P. O.; Cossi, M.; Schimmelpfennig, S.;
Neogrady, P.; Seijo, L. Comput. Mater. Sci. 2003, 28, 222-239.
(41) Azizi, N.; Saidi, M. R. Org. Lett. 2005, 7, 3649-3651.
(42) Raczyꢀska, E. D.; Kosiꢀska, W.; Osꢁ iałowski, B.; Gawinecki, R.
Chem. Rev. 2005, 105, 3561−3612.
1
2
3
4
5
6
7
8
9
(29) Moad, G.; Solomon, D. H. The chemistry of radical polymerization.
Elsevier, 2005.
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
(43) Gutiꢂrrez-Arzaluz, L.; Cortꢂs-Guzmꢃn, F.; Rocha-Rinza, T.; Peꢄn, J.
Phys. Chem. Chem. Phys. 2015, 17, 31608−31612.
(44) Nishina, N.; Mutai, T.; Aihara, J. J. Phys. Chem. A 2017, 121,
151−161.
(45) Henderson, L. M.; Chappell, J. B. Eur. J. Biochem. 1993, 217, 973-
980.
(46) DeRosa, M. C.; Crutchley, R. J. Coordin. Chem. Rev. 2002, 233, 351-
371.
(47) Wrona, M.; Patel, K.; Wardman, P. Free Radical Bio. Med. 2005, 38,
262-270.
(48) Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano, T.
J. Biol. Chem. 2003, 278, 3170-3175.
(49) Liu, Y.; Imlay, J. A. Science 2013, 339, 1210-1213.
(50) Nguyen, D.; Joshi-Datar, A.; Lepine, F.; Bauerle, E.; Olakanmi, O.;
Beer, K.; McKay, G.; Siehnel, R.; Schafhauser, J.; Wang, Y.; Britigan,
B. E.; Singh, P. K. Science 2011, 334, 982-986.
(51) Wasil, M.; Halliwell, B.; Grootveld, M.; Moorhouse, C. P.; Hutchison,
D. C.; Baum, H. Biochem. J. 1987, 243, 867-870.
(52) Wang, W.-F.; Schuchmann, M. N.; Schuchmann, H. P.; Knolle, W.;
Sonntag, J. V.; Sonntag, C. V. J. Am. Chem. Soc. 1999, 121, 238-245.
(53) Verhasselt, S.; Roman, B. I.; Bracke, M. E.; Stevens., C. V. Eur. J.
Med. Chem. 2017, 136, 85–103.
(54) Eberhardt, M. K.; Colina, R. J. Org. Chem. 1988, 53, 1071–1074.
(55) Crank, G.; Mursyidi, A. J. Photochem. Photobiol. A Chem. 1992, 64,
263-271.
(56) Lam, M.; Oleinick, N. L.; Nieminen, A. L. J. Bio. Chem. 2001, 276,
47379-47386.
(57) Kessel, D.; Luo, Y. Cell Death Differ. 1999, 6, 28-35.
(30) Xu, G.; Chance, M. R. Chem. Rev. 2007, 107, 3514-3543.
(31) Zhu, B. Z.; Mao, L.; Huang, C. H.; Qin, H.; Fan, R. M.;
Kalyanaraman, H.; Zhu, J. G. Proc. Natl. Acad. Sci. 2012, 109, 16046-
16051.
(32) Feng, G.; Cheng, P.; Yan, W.; Boronat, M.; Li, X.; Su, J. H.; Wang,
J.; Li, Y.; Corma, A.; Xu, R.; Yu, J. Science 2016, 351, 1188-1191.
(33) Hu, J. J.; Wong, N. K.; Ye, S.; Chen, X.; Lu, M. Y.; Zhao, A. Q.; Guo,
Y.; Ma, A. C.; Leung, A. Y.; Shen. J.; Yang, D. J. Am. Chem. Soc. 2015,
137, 6837-6843.
(34) Bai, X.; Huang, Y.; Lu, M.; Yang, D. Angew. Chem. Int. Ed. 2017,
56, 12873-12877.
(35) Li, M.-D.; Ma, J.; Su, T.; Liu, M.; Yu, L.; Phillips, D. L. J. Phys.
Chem. B 2012, 116, 5882-5887.
(36) Du, L.; Qiu, Y.; Lan, X.; Zhu, R.; Phillips, D. L.; Li, M. D.; Dutton,
A. S.; Winter, A. H. J. Am. Chem. Soc. 2017, 139, 15054−15059.
(37) Dai, J.; Han, J.; Chen, X.; Fang, W.; Ma, J.; Phillips, D. L. Phys.
Chem. Chem. Phys. 2015, 17, 27001-27010.
(38) Xu, Y. C.; Chen, X.; Fang, W. H.; Phillips, D. L. Org. Lett. 2011, 13,
5472-5475.
(39) Gaussian 03, Revision D. Frisch, M. J.; Trucks, G. W.; Schlegel, H.
B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, Jr.,
J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar,
S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.;
Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota,
K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.;
Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.;
Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.;
12
ACS Paragon Plus Environment

Page 13 of 13
Journal of the American Chemical Society
Table of Contents
1
2
3
4
5
6
7
Dynamics of oxygen-independent photocleavage of blebbistatin as a
one-photon blue or two-photon near infrared light-gated hydroxyl
radical photocage
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
H2O
•OH
Blebbistatin
Skin cancer cells
Enhanced cytotoxicity
13
ACS Paragon Plus Environment