Specific Imaging of Tyrosinase in Vivo with 3-Hydroxybenzyl Caged D -Luciferins

Article abstract of DOI:10.1021/acs.analchem.8b01874

Tyrosinase (TYR), a key enzyme in biosynthesis of melanin, usually functions as a biomarker of severe skin diseases such as vitiligo and melanoma cancer. Accurate detection of TYR activity in vivo is urgent but still challenging. Inspired by the advantages of bioluminescence in vivo strategy in imaging and the specific hydroxylation of 3-hydroxybenzyloxy group by TYR, a bioluminogenic probe, TYR-LH₂, was designed and synthesized through caging D-luciferin with 3-hydroxybenzyl. The probe exhibits high selectivity and sensitivity toward TYR with a detection limit of 0.11 U/mL in a small detection volume of 100 μL. Bioluminescence imaging results show that TYR-LH₂ is fully competent for monitoring the dynamic changes of TYR in living cells and model animals and possesses the capability of discriminating melanocytes from other cell lines, thus offering a promising approach for investigation and diagnosis of melanoma cancer and other TYR-related diseases in vivo.

Full text of DOI:10.1021/acs.analchem.8b01874

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Article
Specific Imaging of Tyrosinase in Vivo
with 3-Hydroxybenzyl Caged D-Luciferins
Shuang Li, Rui Hu, Shuangqing Wang, Xudong Guo, Yi Zeng, Yi Li, and Guoqiang Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01874 • Publication Date (Web): 26 Jun 2018
Downloaded from http://pubs.acs.org on June 26, 2018
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Analytical Chemistry
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Specific Imaging of Tyrosinase in Vivo with 3-Hydroxybenzyl Caged
D-Luciferins
Shuang Li,1,3 Rui Hu,*1 Shuangqing Wang,1 Xudong Guo,1 Yi Zeng,2,3 Yi Li,2,3 and Guoqiang Yang*1,3
1Key laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
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2Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry,
Chinese Academy of Sciences, Beijing 100190, China
3University of Chinese Academy of Sciences, Beijing 100049, China
ABSTRACT: Tyrosinase (TYR), a key enzyme in biosynthesis of melanin, usually functions as a biomarker of severe skin diseasꢀ
es such as vitiligo and melanoma cancer. Accurate detection of TYR activity in vivo is urgent but still challenging. Inspired by the
advantages of bioluminescence in vivo strategy in imaging and the specific hydroxylation of 3ꢀhydroxybenzyloxy group by TYR, a
bioluminogenic probe, TYRꢀLH2 was designed and synthesized through caging Dꢀluciferin with 3ꢀhydroxybenzyl. The probe exꢀ
hibits high selectivity and sensitivity towards TYR with a detection limit of 0.11 U/mL in a small detection volume of 100 ꢁL. Bioꢀ
luminescence imaging results show that TYRꢀLH2 is fully competent for monitoring the dynamic changes of TYR in living cells
and model animals and possesses the capability of discriminating melanocytes from other cell lines, thus offering a promising apꢀ
proach for investigation and diagnosis of melanoma cancer and other TYR ꢀrelated diseases in vivo.
Tyrosinase (TYR), a multifunctional, glycosylated and copꢀ
perꢀcontaining oxidase widely distributed in bacteria, plants
and mammals, plays key roles in tyrosineꢀrelated physiologiꢀ
cal processes such as plants browning reaction, insect cuticle
sclerotization, oxidative stress, mammalian melanogenesis and
so on.1ꢀ2 In the presence of molecular oxygen, TYR catalyzes
the orthoꢀhydroxylation of monophenols to oꢀdiphenols and
the further oxidation of oꢀdiphenols to oꢀquinones with which
melanin synthesis in melanocytes is initiated.3ꢀ7 TYR activiꢀ
ty/level directly affect melanin production, which is associated
with some severe skin afflictions including vitiligo and melaꢀ
noma cancer.8 Accordingly, TYR is regarded as a biomarker
and therapeutic target of related diseases and its overexpresꢀ
sion in melanoma cells has motivated the development of
chemical tools to probe this enzyme in living systems.9ꢀ10 Moꢀ
lecular imaging based on fluorescence probes is one of the
most widespread strategies in biosensing due to its high speciꢀ
ficity, sensitivity and noninvasiveness properties.11ꢀ12 Many
TYR fluorescence probes using phenol as the receptor based
on the TYRꢀcatalyzed oxidation of phenols to quinones, have
been synthesized.13ꢀ16 However, fluorescence imaging techꢀ
nique, requiring an external excitation source, is not suited for
some biochemical analysis in living animals, where delivery
of excitation light is often inefficient or impractical.17 Moreoꢀ
ver, fluorescence probes usually present low tissue penetrabilꢀ
ity and high cytotoxicity. These all impede further applications
of fluorescence imaging in vivo. Bioluminescence technique,
taking a native light emission produced by luciferaseꢀ
catalyzed reaction as reporting signal, tactfully avoids the efꢀ
fects of external illumination, and then provides a versatile
platform for imaging living subjects.18 Among the discovered
bioluminescence systems, the firefly luciferinꢀluciferase pair is
the most widely used.19 At the heart of this system is Dꢀfirefly
luciferin that undergoes transformation catalyzed by luciferase
to related oxyluciferin in the presence of adenosine triphosꢀ
phate (ATP), Mg2+ and oxygen, accompanied with emission
out. During the enzymeꢀsubstrate combination, the 6’ꢀ
hydroxy/amino moiety of luciferin is essential.20 Caging at the
6’ꢀ position of luciferin can prevent this combination and then
cause the bioluminescence silence. Once Dꢀluciferin is reꢀ
leased from its caged state, the bioluminescence instantly reꢀ
21
covers. By virtue of this turnꢀon phenomenon, many bioluꢀ
minogenic probes for a myriad of bioꢀanalytes and bioꢀ
processes have been developed, such as βꢀlactamase,22 γꢀ
gultamyltranspeptidase,23 hydrogen peroxide,24 and hypoꢀ
chlorite25 et al, which all exhibit strong ability of interrogation
of cellular and molecular features in vivo.
In 2016, Tung's group reported a TYR bioluminogenic
probe based on aminoluciferin with 4ꢀhydroxybenzyl as its
recognition unit.26 However, TYR probes bearing 4ꢀ
hydroxybenzyl as their recognition moiety always suffer false
responses from other reactive oxygen species (ROS) such as
H2O2, ONOOꢀ and HClO. In contrast, 3ꢀhydroxybenzyl group,
a recently developed TYR recognition moiety, specifically
facilitates its hydroxylation by TYR, ruling out the interferꢀ
ences of ROS.27 Combining this new TYR recognition moiety
and bioluminescence imaging (BLI) technique, a new bioluꢀ
minogenic probe (TYRꢀLH2) was designed and synthesized in
this work by straightforward modification of
a
3ꢀ
hydroxybenzyl group at the 6’ꢀposition of Dꢀluciferin. Its
principle for TYR detection is depicted in Scheme 1. Hydroxꢀ
ylation of TYRꢀLH2 catalyzed by TYR triggers the cleavage of
the connection between 3ꢀhydroxybenzyl and Dꢀluciferin
group, resulting in the release of Dꢀluciferin, which is subseꢀ
quently oxidized in the present of luciferase and ATP, giving a
bright bioluminescence emission for signaling TYR level in
turn. The bioluminogenic probe TYRꢀLH2 shows high selecꢀ
tivity and sensitivity towards TYR in vitro as well as in vivo,
offering a new accurate tool for evaluation of the TYR activiꢀ
ty/level and its related diseases especially malignant melanoꢀ
ma in model animals.
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neously implanted under the right rear thigh of each fourꢀweek
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old (weight of 18 g) BALB/c nude mouse. Mice were housed
for about 2 weeks till the tumors grew to 1.0 cm in diameter,
then divided into 2 groups (n = 3 for each group). Mice in
group 1 were intraperitoneally injected with 100 ꢁL of probe
(1 mM) each. Mice in group 2 were treated with kojic acid
(100 ꢁL, 200 ꢁM) 1 hour before the injection of the probe and
used for negative control test. Bioluminescent images were
acquired with an acquisition time of 10 min. Image processing
was performed using imaging software (Living image R4.4).
ROI was selected to cover the whole tumor. All luminescent
intensity values were normalized by the luminescence intensiꢀ
ty before the injection of TYRꢀLH2.
Scheme 1. Illustration of TYRꢀLH2 structure and its bioluminesꢀ
cence detection mechanism for TYR.
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EXPERIMENTAL SECTION
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General Information. Chemicals were purchased from
commercial sources, supplied by J&K Chemical and Beijing
InnoChem, and used without further purification. Bioreagents,
tyrosinase, GGT, leucin aminopeptidase and luciferase from
Photinus pyralis (L9506) were obtained from SigmaꢀAldrich.
The cell lines were obtained from JENNIO BioTECH Co.
Ltd., Guangzhou, China. Fluorescence spectra were recorded
on Hitachi Fꢀ7000. MTT assays were carried out on a BioTek
microplate reader, 1H NMR spectra were obtained from
Bruker DMXꢀ300 and DMXꢀ400 spectrometers. Electrospray
ionization mass spectra (ESIꢀMS) were measured out with a
Shimadzu LCꢀMS 2010A instrument. Bioluminescence intenꢀ
sity were measured on an EnVision 2103 Multilabel Reader
(Perkin Elmer). Bioluminescence images in vitro, cells and
vivo were taken with a Xenogen IVIS Spectrum system (Caliꢀ
per Life Sciences, Hopkinton, MA, USA). Images taken with
the IVIS Spectrum were analyzed with Living Image R4.4
software.
RESULTS AND DISSCUSSION
Bioluminescence Response of TYR-LH2 to TYR in Vitro.
The bioluminescence response of TYRꢀLH2 towards TYR in
vitro was first investigated by recording the signal intensity at
570 nm at different pH, temperature and incubation time with
and without TYR. Almost no emission signal could be detectꢀ
ed from TYRꢀLH2 sample in the absence of TYR by addition
of luciferase and ATP at pH ranging from 6.0 to 9.0 and temꢀ
perature from 25 to 45 °C, as shown in Figure S1. When coꢀ
incubating with TYR, strong emission from TYRꢀLH2 sample
was observed with signal maximums at pH 7.4 and 37 °C,
respectively, which are exactly coincident with the physiologiꢀ
cal conditions. The emission profile of the incubation buffer
with TYRꢀLH2 and TYR (Figure S2) is identical to that of free
luciferin, indicating that the bioluminogenic probe TYRꢀLH2
can be triggered by TYR to release luciferin. ESIꢀMS study
further strengthened such catalytic production by observing
the molecular ion peak of luciferin at m/z 279.0. To further
assess the bioluminescence response of TYRꢀLH2 towards
TYR, the timeꢀdependent emission of TYRꢀLH2 with TYR of
several concentrations were measured respectively, and the
results are depicted in Figure 1. The signal intensities derived
from TYRꢀLH2 with addition of TYR increased steeply at the
incubation time from 0 to 50 min, and then reached the plateau
and lasted for the remaining detection time of 190 min. Conꢀ
trol sample without TYR showed no bioluminescence signal,
indicating no luciferin released during the 4 h incubation. The
incubation time and TYR concentration courses reveal that the
bioluminescence response of TYRꢀLH2 towards TYR are both
time and concentration dependent, and more than 300ꢀfold
enhancement of bioluminescence response was detected after
3 h incubation with 200 U/mL TYR at physiological condiꢀ
tions. These conditions were also adopted in the following
assays.
Synthesis. Probe TYRꢀLH2 was synthesized by a facile twoꢀ
step combination from commercial materials as shown in
Scheme S1. Under argon atmosphere, a solution of 3ꢀ
hydroxybenzyl bromide was dropwise mixed with 2ꢀcyanoꢀ6ꢀ
hydroxybenzothiazole in the presence of K2CO3 and acetoniꢀ
trile to obtain intermediate 1 after flash column chromatogꢀ
raphy separation. Then the condensation reaction of 1 and Dꢀ
cysteine hydrochloride followed by recrystallization gave the
pure final product of TYRꢀLH2
In Vitro Bioluminescence Measurements. The in vitro
measurement assays were all performed in PBS buffer (pH =
7.4) with 10 mM MgSO4 at 37°C. 50 ꢁL buffer solutions with
various enzyme concentrations (0–200 U/mL) were added into
TYRꢀLH2 solutions (20 ꢁM, 50 ꢁL) and then incubated for 3 h.
To the subsequent solutions, 50 ꢁL buffer solution of firefly
luciferase (20 ꢁg/mL) containing 2.6 mM ATP was added.
The bioluminescent signals were taken after a second incubaꢀ
tion period of 5 min.
Cell Bioluminescence Imaging. Cell lines were passed and
planted in 96ꢀwell plate (Corning, 3603) at 37 °C under 5%
CO2 atmosphere for 24 h before imaging. During the TYR
activity assay, 100 ꢁL of TYRꢀLH2 with various concentraꢀ
tions (0 ꢀ 35 ꢁM) in FBSꢀfree DMEM solution were added
into the culture wells in which the medium had been discarded
before. After 60 min incubation and 5 min exposure, cell imꢀ
ages were taken using a Xenogen IVIS Spectrum system. In
the TYR inhibition assay, cell lines were first treated with 50
ꢁL kojic acid of different concentrations (0, 150 and 200 ꢁM)
for 1 h, respectively, then added with 100 ꢁL of TYRꢀ
LH2/DMEM. After incubation for another 1 h and exposure
for 5 min, the bioluminescence signal was measured.
In Vivo Bioluminescence Imaging. To generate tumor
xenografts in mice, B16ꢀluc cells (1 × 107 cells) were subcutaꢀ
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Figure 1. Plots of the bioluminescence intensity versus the incubaꢀ
tion time of TYRꢀLH2 (20 µM) with TYR of several concentraꢀ
tions in PBS buffer (pH = 7.4) at 37 °C.
TYR for 1 h, and then TYRꢀLH2 was added for further incubaꢀ
tion with 3 h. After lightingꢀup the mixture by luciferase and
ATP, signals were recorded and
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Figure 2. Doseꢀdependent bioluminescence image and its correꢀ
sponding relative bioluminescence intensity of TYRꢀLH2 (20 ꢁM)
towards TYR of different concentrations (0, 5, 10, 20, 50, 150,
200 U/mL) in PBS buffer, pH = 7.4, 37 °C; (insert: plot of the
relative bioluminescent intensity as a function of TYR concentraꢀ
tion and the linear fitting result). Signals were normalized to the
signal from TYRꢀLH2 without TYR and expressed as the means ±
standard deviation (S.D.) of four independent measurements.
To quantify the emission response of TYRꢀLH2 towards
TYR, a doseꢀdependent assay of TYRꢀLH2 (20 ꢁM) against
TYR (0–200 U/mL) was conducted under 3 h incubation. As
shown in Figure 2, the bioluminescence signal from TYRꢀLH2
increases proportionally with the concentration of TYR. The
plot of luminescence intensity versus TYR concentration was
well linearly fitted at the concentration range of 0–200 U/mL.
This tendency continued down to the very low concentration
range of TYR (0–1.0 U/mL, Figure S3) and the detection limit
was accordingly calculated as low as 0.11 U/mL (3σ criterion),
that is the lowest value detected in such a small volume (≤ 100
ꢁL) which may forebode the successful application of TYR
imaging in cells.
The bioluminescence responses of TYRꢀLH2 to some potenꢀ
tial bioꢀinterferents were further examined, including ROS
−
(H2O2, ClO−, tertꢀbutyl hydroperoxide (TBHP), ·OH, O2 ,
ONOO−), reductive species (vitamin C) and other common
existing biological substances (glutathione, cysteine, glycine,
glucose, human serum albumin) and enzymes (GGT, leucine
aminopeptidase, monoamine oxidase A). TYRꢀLH2 was incuꢀ
bated with these selected bioꢀspecies and TYR, respectively,
followed by addition of the same amount of luciferase and
ATP. The bioluminescence results are shown in Figure 3A and
3B. Compared with TYR, negligible signal enhancement was
observed after 3 h incubation with all the potential bioꢀ
interferents, indicative of the high selectivity of TYRꢀLH2
towards TYR. The selectivity of TYRꢀLH2 to TYR may be
ascribed to the specific hydroxylation of 3ꢀhydroxybenzyl
group by TYR and the subsequent 1,6ꢀrearrangement–
elimination reaction27, by which free Dꢀluciferin is released
and then lightedꢀup by luciferase and ATP. A TYR activity
inhibition assay was meanwhile performed to strengthen the
specific recognition of TYRꢀLH2 towards TYR. Kojic acid (0–
500 ꢁM), a standard inhibitor of TYR, was preꢀcultured with
Figure 3. (A). Bioluminescence image and the bioluminescence
–
response of TYRꢀLH2 (20 ꢁM) to ROS (H2O2, OONO–, ClO–, O2
, .OH, NO, NO2– and TBHP; 100 ꢁM each) and TYR (200 U/ml);
(B) Bioluminescence image and the bioluminescence response of
TYRꢀLH2 (20 ꢁM) to bioꢀactive substances including glucose (1.0
mM), glycine (1.0 mM), glutathione (GSH, 1.0 mM), cysteine
(Cys, 1.0 mM), leucine aminopeptidase (LAPs, 25 U/L), human
serum albumin (HAS, 100 ꢁM), monoamine oxidase A (MAO, 25
U/L), GGT (20 U/L) and TYR (200 U/mL). Signals were all norꢀ
malized to the signal from TYRꢀLH2 in the absence of tyrosinase
expressed as means ± standard deviation of four separate measꢀ
urements; (C) Bioluminescence image and the corresponding
relative intensity of TYRꢀLH2 (20 ꢁM) with TYR mixed kojic
acid of various concentrations (0, 1, 5, 10, 50, 100, 150, 200, 500
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ꢁM). Signals were normalized to the signal from TYRꢀLH2 treatꢀ
ed with TYR. Error bars represent ± S.D (n = 4).
for 1 h. Signals were normalized to signal from B16ꢀluc cells
incubated with TYRꢀLH2 without preꢀtreated by kojic acid. Error
bars represent ±S.D. (n = 4).
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depicted in Figure 3C. The existence of kojic acid led to draꢀ
matic decrease of bioluminescence intensity. The luminesꢀ
cence intensity changed in inverse proportion to the concentraꢀ
tion of kojic acid and less than 10% signal remained when the
concentration of kojic acid reached to 500 ꢁM, suggesting that
the bioluminogenic probe TYRꢀLH2 was a specific substrate
of TYR.
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Bioluminescence Imaging of TYR in Cells and in Vivo.
Three luciferaseꢀexpressing cell lines, murine melanoma B16ꢀ
luc cells with TYR overexpression as target model, Helaꢀluc
and NIH/3T3ꢀluc cells with normal TYR expression as control
models, were used to demonstrate the potential of TYRꢀLH2
for detecting TYR in living cells. Biocompatibility is prerequiꢀ
site for probes used in living cells, therefore a standard 3ꢀ(4,5ꢀ
dimethylꢀ2ꢀthiazolyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT)
assay was first conducted to evaluate the cytotoxicity of TYRꢀ
LH2 on these cell lines. Even at a high concentration of 35 ꢁM,
over 80% of cells were still survived as shown in Figure S4,
indicative of good compatibility of TYRꢀLH2 to model cell
lines. Then the three cell lines were incubated with TYRꢀLH2
of different concentrations (0 – 35 ꢁM) at 37 for 60 min and
monitored by cell imaging, giving the bioluminescence results
depicted in Figure 4A. In contrast to the dark signal in the
three cells without TYRꢀLH2, bioluminescence burst and inꢀ
creased linearly with the concentration of TYRꢀLH2, suggestꢀ
ing that the catalytic hydroxylation as well as the resulting Dꢀ
luciferin
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Figure 5. (A) Timeꢀdependent bioluminescence imaging of mice
xenografted with Lucꢀtransfected B16 cells tumors after intraperiꢀ
toneal injection of 100 ꢁL of TYRꢀLH2(1 ꢁM), top row; images of
mice treated with 100 ꢁL of kojic acid (200 ꢁM) 1h later followed
by 100 ꢁL of TYRꢀLH2 (1 ꢁM) injected, bottom row; (B) Total
flux of (A). Error bars were represent ±S.D (n = 3).
release occurred in these living cells and both TYR and lucifꢀ
erase were not saturated with TYRꢀLH2 up to 35 ꢁM. Howevꢀ
er, the bioluminescence intensity in the three cell lines was
very different. Emission signal appeared in B16ꢀluc cells was
2ꢀ and 3ꢀ fold stronger than that in Helaꢀluc cells and in
NIH/3T3ꢀluc cells, respectively, which is attributed to the difꢀ
ferent extent of TYR expression in different cells. To verify
this causality, kojic acid of different concentrations (0, 150
and 200 ꢁM) was used to pretreat B16ꢀLuc cell lines before
incubation with probe TYRꢀ LH2. As shown in Figure 4B, the
emission signal showed evident trend of suppression and more
than a half decrease after treated with kojic acid of 200 ꢁM.
These results demonstrated that the bioluminogenic probe
TYRꢀLH2 can be used to image endogenous TYR activity in
living cells, even further to selectively discriminate melanoma
cell lines.
The performance of TYRꢀLH2 for melanoma visualization
in living mice was examined. Nude mice with xenografted
tumor of B16ꢀluc cells in the right thigh were randomly used
in two groups ‒ with and without preꢀinjected kojic acid. After
TYRꢀLH2 (1.0 ꢁM, 100 ꢁL) were intraperitoneal (i.p.) injectꢀ
ed, light production was monitored at every 10 min interval
for a period of 140 min. The time courses of emission from
TYRꢀLH2 in vivo are depicted in Figure 5 and Figure S5. Disꢀ
tinct emission signal from tumor regions in mice was observed
after the first 10 min interval, suggesting good tissue penetraꢀ
tion and sensitive response of probe TYRꢀLH2. During the 140
min monitoring period, emission signal underwent an “inꢀ
crease–maintain–decrease” course with an intensity peak at 70
min. The long lighting duration provides potential advantages
for observation and related bioꢀoperations. A similar time
course of bioluminescence signals but 2.3ꢀfold lower in intenꢀ
sity were obtained for mice preꢀtreated with kojic acid showꢀ
ing a clear inhibition effect. These imaging results demonꢀ
Figure 4. (A) Total photon flux of bioluminescence responses of
B16ꢀluc, Helaꢀluc and NIH/3T3ꢀluc incubated with TYRꢀLH2 of
various concentrations (0ꢀ35 ꢁM) at 37 °C for 3 h; (B) Biolumiꢀ
nescence images and the normalized bioluminescence intensity of
B16ꢀluc cells pretreated with 0, 150 and 200 ꢁM kojic acid, reꢀ
spectively for 1 h and incubated with TYRꢀLH2 (20 ꢁM) at 37 °C
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5.
Mirica, L. M.; Vance, M.; Rudd, D. J.; Hedman, B.; Hodgꢀ
strate that TYRꢀLH2 has the potential to be used to dynamicalꢀ
ly visualize the TYR activity in model animals.
son, K. O.; Solomon, E. I.; Stack, T. D. P. Science 2005, 308 (5730),
1890ꢀ1892.
1
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6.
7.
Lin, J. Y.; Fisher, D. E. Nature 2007, 445 (7130), 843ꢀ850.
Chai, L. J.; Zhou, J.; Feng, H.; Tang, C.; Huang, Y. Y.; Qiꢀ
CONCLUSION.
an, Z. S. Acs Appl. Mater. Inter. 2015, 7 (42), 23564ꢀ23574.
8. Han, R.; Baden, H. P.; Brissette, J. L.; Weiner, L. Pigm.
Cell Res. 2002, 15 (4), 290ꢀ297.
In summary, a new TYR bioluminogenic probe TYRꢀLH2 by
introducing a 3ꢀhydroxybenzyl group into Dꢀluciferin was
prepared and its property for TYR imaging in vitro and in vivo
were subsequently evaluated. The specific hydroxylation of 3ꢀ
hydroxybenzyl group by TYR combined with the clean backꢀ
ground of bioluminescence strategy endows this probe high
selectivity and sensitivity towards TYR. A detection limit of
0.11 ꢁM was obtained in a small detection volume of 100 ꢁL
which encouraged the applications of TYRꢀLH2 in vivo. Imagꢀ
ings from living cells and model animals showed that TYRꢀ
LH2 presented capability of identifying the dynamic changes
of TYR as well as discrimination of melanocytes from other
cell lines, affording new opportunities for investigation and
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ASSOCIATED CONTENT
Supporting Information
Steinhardt, R. C.; O'Neill, J. M.; Rathbun, C. M.;
The Supporting Information is available free of charge on the
ACS Publications website.
Prescher, J. A.; Contag, C. H. Curr. Opin. Chem. Biol.
Vieira, J.; da Silva, L. P.; da Silva, J. C. G. E. J. Photo-
AUTHOR INFORMATION
Corresponding Author
Mcelroy, W. D.; Seliger, H. H.; White, E. H. Photochem.
* Eꢀmail: hurui@iccas.ac.cn;
* Eꢀmail: gqyang@iccas.ac.cn
Li, J.; Chen, L. Z.; Du, L. P.; Li, M. Y. Chem. Soc. Rev.
Yao, H.; So, M. K.; Rao, J. Angew. Chem. Int. Edit. 2007,
Notes
The authors declare no competing financial interest. A
Li, S.; Hu, R.; Yang, C. L.; Zhang, X.; Zeng, Y.; Wang, S.
ACKNOWLEDGMENT
This work was supported by the National Natural Science Founꢀ
dation of China (21233011) and Youth Innovation Promotion
Association (2017032) of Chinese Academy of Sciences.
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