External-Radiation-Induced Local Hydroxylation Enables Remote Release of Functional Molecules in Tumors

Article abstract of DOI:10.1002/anie.202005612

Radiation-induced cleavage for controlled release in vivo is yet to be established. We demonstrate the use of 3,5-dihydroxybenzyl carbamate (DHBC) as a masking group that is selectively and efficiently removed by external radiation in vitro and in vivo. D

Full text of DOI:10.1002/anie.202005612

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: External Radiation–Induced Local Hydroxylation Enables Remote
Release of Functional Molecules in Tumors
Authors: Qunfeng Fu, Hongyu Li, Changlun Wang, Siyong Shen,
Huimin Ma, and Zhibo Liu
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202005612
Link to VoR: https://doi.org/10.1002/anie.202005612

10.1002/anie.202005612
Angewandte Chemie International Edition
COMMUNICATION
External Radiation–Induced Local Hydroxylation Enables Remote
Release of Functional Molecules in Tumors
Qunfeng Fu,[a] Hongyu Li,[c,d] Dongban Duan,[a] Changlun Wang,[a] Siyong Shen,[a] Huimin Ma[c,d] and
Zhibo Liu*[a,b]
Abstract: Radiation-cleavage chemistry that can achieve controlled
release of functional molecules in vivo is of high clinical relevance yet
has not been established. Here, we describe 3,5-dihydroxybenzyl
carbamate (DHBC) as a masking group that can be selectively and
efficiently removed by external radiation in vitro and in vivo. DHBC
reacts mainly with hydroxyl radical among reactive oxygen species
produced by radiation to afford hydroxylation on para/ortho positions,
followed by 1,4- or 1,6-elimination to rescue the diverse functionality
of the client molecule. We found that the reaction is rapid and can
liberate client functional molecules under physiological conditions.
This controlled release platform is compatible with living organisms,
as demonstrated by the release of a rhodol fluorophore derivative (O-
methylrhodol, MeRho) in three cancer cell lines and mouse tumor
xenografts. The combined benefits from the robust caging group, the
good release yield and the independence of penetration depth make
DHBC derivatives attractive chemical caging moieties for use in
radiation-perturbing chemical biology and prodrug activation in vivo.
radiation can be a transformed light source to control cleavage
chemistry in vivo, especially in a tumor-selective manner.
Ionized
H2O Radiation
e-aq, H+, H•, •OH, H2, H2O2
2.63
0.55 2.72 0.45 0.68
Tumor
Body
DHBC-MeRho
MeRho
Scheme 1. A radiation–induced cleavage chemistry that could remotely release
small molecules in the tumor. In the box, we showed the design of radiation-
induced hydroxylation of the 3,5-dihydroxylbenzyl carbamate-linked MeRho for
releasing MeRho. Fluorescence of MeRho is quenched in DHBC-MeRho;
radiation-induced “cleavage” of the linker releases fluorescent MeRho.
On the clinical side, radiotherapy (RT) is widely used as a
first-line treatment in oncology for localized cancer or isolated
metastasis[10]
. However, radiotherapy, as a monotherapy,
generally fails to cure cancers completely since its efficacy can be
attenuated by many factors. To enhance therapeutic effects and
prevent the recurrence of cancer, multimodal therapies, such as
Cleavage chemistry that can operate in vivo with high
temporal-spatial resolution is greatly needed for both biological
research[1] and clinical use[2] but is still a formidable challenge[3].
Light-induced photocleavage chemistry is capable of precisely
regulating protein function in cultured cells[4] or controlling prodrug
activation in the outermost layer of the body[5], but the limitation of
tissue penetration hampers its application in living systems[3].
Emerging strategies based on two-photon fluorescence[6] or
upconversion nanoparticles[7] have partially solved this problem,
yet the penetration depth is generally less than 3 mm[6-8]. Notably,
tissue penetration of radiation, including X-rays, γ-rays, and high-
energy particle rays (i.e., electron, proton, α-particle, etc.), is
remarkably deeper[8-9] (up to 15 cm) and can be readily guided by
high-resolution computed tomography (CT) or magnetic
resonance imaging (MRI). In this matter, we wondered whether
concurrent chemoradiotherapy (CRT), are widely used[11]
.
However, patients treated with chemoradiotherapy suffer from a
range of side effects from normal chemotherapy, as it is often a
simple concurrent or sequential injection of chemotherapy drugs
after radiotherapy. One approach aimed at improving the
selectivity of tumor cell killing is to use less toxic prodrug forms
that can be selectively activated in tumor tissues[12]. The
aforementioned radiation-cleavage chemistry, if successfully
developed, can be applied to remove the masking group directly
and thereby provide an ideal means to perform radiation-guided
chemotherapy in a tumor-selective manner.
External light suffers from rapid attenuation through tissue
and therefore cannot reach malignant lesions hidden under deep
tissue. A recent approach that utilizes Cerenkov radiation as an
internal light source has shed light on overcoming the limitation of
tissue penetration[13]. Cerenkov radiation (CR) happens when
charged particles, such as β+ and β-, travel through a dielectric
medium beyond the speed of light. The emission spectrum is
continuous and includes ultraviolet (UV) and visible light[14]. The
dominant emission of CR is UV, which is ideal for UV responsive
photocleavable groups (e.g., ortho-nitrobenzene)[15] and
photosensitizers (e.g., TiO2 and porphyrin)[16]. Previously, we
reported the successful use of Ga-68 as a Cerenkov light source
[a]
Q. F. Fu, D. B. Duan, C. L. Wang, S. Y. Shen, Prof. Dr. Z. B. Liu
College of Chemistry and Molecular Engineering, Peking University,
Beijing, 100871 (China)
Radiation Chemistry Key Laboratory of Fundamental Science,
Beijing National Laboratory for Molecular Sciences, E-mail:
zbliu@pku.edu.cn.
[b]
[c]
Prof. Dr. Z. B. Liu
Peking University-Tsinghua University Center for Life Sciences,
Beijing, 100871 (China)
Dr. H. Y. Li and Prof. Dr. H. M. Ma
to activate TiO2 for depth-independent photodynamic therapy[16]
.
Beijing National Laboratory for Molecular Sciences, Key Laboratory
of Analytical Chemistry for Living Biosystems, Institute of Chemistry,
Chinese Academy of Sciences
To test whether Ga-68 could serve as a Cerenkov light source to
induce cleavage chemistry, photocleavable compounds 2–9 were
synthesized and characterized (Figure 1A) that can release
Fmoc-lysine under UV irradiation[17]. Compound 1 was prepared
and tested as a negative control. Stock solutions of 10 µM
compounds 1–9 were prepared in PB (0.02 M phosphate buffer,
pH=7.4) and then incubated with 8.0 mCi of Ga-68 overnight. The
crude reaction was analyzed by liquid chromatography–mass
spectrometry (LC-MS) to determine the amount of released
Beijing 100190 (China)
[d]
Dr. H. Y. Li and Prof. Dr. H. M. Ma
University of the Chinese Academy of Sciences,Beijing 100049
(China)
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.202005612
Angewandte Chemie International Edition
COMMUNICATION
Fmoc-lysine and the stability of the precursor at physiological pH.
The experiment was repeated independently 3 times, and the
results are summarized in Figure 1B. It was found that the Fmoc-
lysine released from compound 2 and compound 5 was
remarkably greater than that of other candidates. To further
investigate whether Cerenkov light was the key factor that
triggered the release, compounds 1–9 were also tested by
irradiation with UV light for 10 min, and the release yield is
summarized in Figure 1C. Contradictory to our previous
observation, the released Fmoc-lysine from compound 2 was
minimal, and compound 5 showed a lower response to UV than
those of compounds 3 and 4. Therefore, we hypothesized that
Cerenkov light is probably not the dominant factor for the
controlled release after incubation with Ga-68.
other compounds were not notably more abundant than the
control group. By carefully analyzing the difference in chemical
structure between compounds 2, 5 and the others, we concluded
that the efficiency of such radiation-cleavage chemistry is highly
correlated to the electron density on the aromatic ring of the
caging moiety.
Fmoc-lysine was then caged with more electron-rich
aromatic rings to give compounds 10 and 11. However, the
stability of both compounds 10 and 11 was poor in PB. We
assume that the decomposition was due to the para-methoxy
group, which could induce a 1,6-elimination reaction to lead to the
subsequent decarbamation. To address this problem, compound
12 was synthesized, and the release efficiency was tested under
the same conditions. As shown in Figure 1E, the amount of Fmoc-
lysine released from compound 12 was 55 times greater than that
of compound 1 and 5 times greater than that of compound 5.
Encouraged by such an improvement, the radiation-responsive
group was further optimized by replacing the methoxy group with
a phenolic hydroxyl group to give compound 13, whose release
efficiency was over 100 times higher than that of compound 1 and
was approximately twice that of compound 12. It is worth noting
that the Fmoc group contains two aromatic rings but did not
significantly compromise the release efficiency, which is indeed
inspiring, thus prompting us to pursue the underlying mechanism
of this radiation-induced cleavage reaction.
As illustrated in Scheme 1, the radiolysis of water through
ionizing radiation results in the production of highly reactive
species, including hydroxyl radicals and hydrated electrons,
etc.[17]. G-value of hydroxyl radicals is 2.72 (G-value means
number of molecules formed by absorbing 100 eV energy in the
system). We assumed that the aromatic ring undergoes an
8 mCi 68Ga
10 min UV
B
C
100
75
50
25
0
100
75
50
25
0
electrophilic substitution with hydroxyl radicals to form phenol[18]
,
followed by a 1,4- or 1,6-elimination reaction to release the caged
molecules (lysine or Fmoc-lysine, Figure S1). To test this
hypothesis, compound 13 was incubated with 10 mM H2O2, t-
BuOH and DMSO during irradiation, which is a classic assay to
10 Gy γ-ray
10 Gy γ-ray
D
E 300
verify
a hydroxyl radical-mediated process. As expected,
100
75
50
25
0
compared with the sample of 10 μM compound 13 in PB, the
amount of released Fmoc-lysine in 10 mM H2O2 increased, as
H2O2 is known to produce extra hydroxyl radicals under irradiation
(Figure S2). In contrast, the addition of 10 mM t-BuOH and DMSO,
200
100
0
which are known as quenchers of hydroxyl radicals[19]
,
significantly reduced the amount of released Fmoc-lysine (Figure
S2). We further verified this •OH-mediated mechanism by
capturing the reaction intermediate. Compound 14 (100 μM in
deionized water) was irradiated with γ-rays (10 Gy/min) for 10 min
(compound 13 was not used here due to poor aqueous solubility).
The LC-MS assay showed that two new UV absorption peaks
appeared in front of compound 14 (Figure S3), and their mass
spectra matched with the molecular weight of the proposed
intermediates (Figure S4 and S5), indicating positive evidence of
hydroxylation. After incubating at 37 °C for 1 h, these new peaks
disappeared (Figure S3), while the peak of free lysine increased
accordingly (Figure S6), which is corroborative to our assumption.
The radiation-induced cleavage of molecule 12 was also assayed
by LC-MS. The removal moieties, including para- and meta-
hydroxylated benzyl alcohol, were captured by mass
spectrometry and then identified by the subsequent injection of
reference compounds (Figure S7). Supporting this notion,
physical chemists have disclosed that •OH is strongly electrophilic
and acts as a sensitive probe of charge distribution on substituted
Figure 1. 3,5-Dihydroxybenzyl carbamate (DHBC) was identified as a masking
group that can be readily removed by radiation. (A) Structures of compounds
that have been assayed by radiation-induced controlled release. Normalized
amount of Fmoc-lysine released from compounds 1–9 (10 μM) in PB (pH=7.4,
0.1% DMF) after treatment with (B) 8 mCi 68Ga incubation, (C) 10 min 365 nm
UV irradiation, and (D)10 Gy of γ radiation. (E) Summary of the concentration
of Fmoc-lysine released from compounds 1–9, 12 and 13 (10 μM) in PB (pH=7.4,
0.1% DMF) after treatment with 10 Gy of γ radiation (Co-60 as the γ radiation
source, 1 Gy/min, 10 min). The radiation dose rate was 1 Gy/min unless
otherwise noted.
To test our hypothesis, the samples of compounds 1–9 were
further tested with Co-60, from which the irradiation is pure γ-ray
without Cerenkov light. The sample solution was irradiated with
10 Gy of γ-rays and then analyzed by LC-MS to determine the
yield with free Fmoc-lysine. Interestingly, the results with Co-60
irradiation were consistent with those incubated with Ga-68
(Figure 1D). Free Fmoc-lysine from compounds 2 and 5 was 10
times more abundant than that of the control group, while the
This article is protected by copyright. All rights reserved.

10.1002/anie.202005612
Angewandte Chemie International Edition
COMMUNICATION
aromatics. Therefore, we concluded that this radiation-induced
cleavage chemistry is a 1,4- or 1,6-elimination reaction initiated
by •OH-mediated hydroxylation.
fluorescence spectra were measured at 518 nm with excitation at 477 nm. (B-
E) Co-60 was used as the source of γ radiation.
DHBC-MeRho is highly stable and showed no spontaneous
hydroxylation even after a 12 h incubation in PB. The selectivity
of DHBC-MeRho to radiation was examined by measuring the
response upon treatment with various reactive oxygen species.
Treating DHBC-MeRho with 100 equiv. of ROS (HOCl, ONOO-,
NO, 1O2, TBHP, and H2O2) and Fe3+ did not cause release of free
MeRho (Figure 2E). Considering that Fe2+ reacts with dissolved
oxygen, it may also generate •OH[20]. We observed a low level of
release by treating DHBC-MeRho with Fe2+. Interestingly, upon
treated with 1 equiv. of Fenton reagent ([H2O2]:[Fe2+]=10:1,
[Fe2+]=10 μM with equimolar of EDTA) and 60 Gy of γ ray, DHBC-
MeRho showed >10-fold and >50-fold increases in fluorescence
intensity, respectively. This is unexpected, because in theory 1
equiv. of Fenton reagent can generate 10 μM •OH, and 60 Gy of
γ-ray can generate 16.8 μM. This result indicates that radiation is
significantly more efficient than Fenton reagent to trigger the
hydroxylation-mediated controlled release. In addition, it would be
difficult to induce hydroxylation with Fenton reagent in vivo, but γ-
ray can afford local generation of •OH with high spatial and
temporal resolution.
We therefore wondered whether radiation-induced cleavage
chemistry could be developed into a useful controlled release
system, making radiation a perturbing tool for in vivo manipulation.
To pursue this idea, we employed DHBC system to construct a
radiation-responsive fluorogenic probe in which the carbamate
carbon is linked to a client MeRho fluorescent molecule (Figure
2A). Hydroxylation of this system will trigger elimination of the
carbamate, leading to the breakdown of the amide bond,
releasing MeRho (Figure S8). Free MeRho, as opposed to
amine-blocked MeRho in the 3,5-dihydroxybenzyl carbamate
system, emits green fluorescence, providing a simple quantitative
assay for radiation-induced release. According to Figure S9 and
Figure S10, DHBC-MeRho and MeRho showed remarkable
differences in their UV absorption and fluorescence emission
spectra. DHBC-MeRho is stable in PB in the pH range from 4.0
to 11.0 (adjusted by HCl or NaOH). The fluorescence intensity of
both DHBC-MeRho and MeRho is pH-independent within the pH
range of 4 to 10 (Figure S11). The radiation-induced fluorescence
release assay of DHBC-MeRho was performed under
physiological conditions. The concentration of DHBC-MeRho was
10 μM, and the radiation dose rate was 1 Gy/min. By controlling
the irradiation time, the total radiation dose was gradient ascent
from 0 to 60 Gy. The fluorescence intensity was found to increase
with the dose of radiation (Figure 2C) in a linear manner (Figure
2D), and the molar number of molecules produced by 1 J of
radiation energy was 16 nM/Gy.
DHBC-MeRho is also highly stable when treated with various
metal ions, amino acids and other possible analytes in cells
(Figure S12). Collectively, DHBC-MeRho showed significantly
high selectivity for •OH over other analytes. Taken together, the
intrinsic stability of the DHBC-containing linkage and its
robust/specific cleavage by radiation suggest great potential for
the controlled release of bioactive molecules in living systems.
A
60 Gy
B
D
C
E
50 Gy
40 Gy
30 Gy
20 Gy
10 Gy
0 Gy
2500
2000
1500
1000
500
0 Gy 10 Gy 20 Gy 40 Gy 60 Gy
0
500
520
540
560
580
600
50 μm
60
50
40
30
20
10
0
Wavelength (nm)
60
0 Gy
5 Gy
10 Gy
20 Gy
40
20
0
B
C
D
4T1
***
***
***
HeLa
MC38
***
***
1 equiv.
***
***
1000
1000
500
0
1000
100 equiv.
***
***
*
*
**
0
10 20 30 40 50 60
Dose (Gy)
500
0
500
0
0
0
0
1
5
10 20
1
5
10 20
1
5
10 20
Figure 2. Radiation-induced hydroxylation can release MeRho from a DHB-
containing carbamate linker in chemical systems. The fluorescence of MeRho
is “caged” in DHBC-MeRho. (A) Schematic representation of radiation-induced
Dose (Gy)
Dose (Gy)
Dose (Gy)
hydroxylation that can release the fluorescence-“caged” MeRho from
a
Figure 3. Radiation-induced hydroxylation can release MeRho from a DHB-
containing carbamate linker in vitro in dose-dependent manner. (A)
designed DHB-containing carbamate linker. (B) Photography of the
fluorescence-“decaging” of radiation-induced hydroxylation of DHBC-MeRho.
(C) Fluorescence emission spectra of DHBC-MeRho (10 μM) upon treatment
with a dose gradient of radiation in PB (pH=7.4, 0.1% DMF) at 25 °C. (D)
Fluorescence intensity of DHBC-MeRho as a function of radiation dose (0-60
Gy). (E) Fluorescence responses of DHBC-MeRho toward various ROS. The
a
Representative confocal fluorescence images of MC38 cells, 4T1 cells, and
HeLa cells. The cells were pretreated with DHBC-MeRho (10 μM in Hank’s
balanced salt solution, pH=7.4, 0.1% DMF) for 30 min followed by different
amounts of radiation. (B-D) Fluorescence in MeRho-positive cells in the
examined field of view by confocal microscopy. Twenty fields of view were
This article is protected by copyright. All rights reserved.

10.1002/anie.202005612
Angewandte Chemie International Edition
COMMUNICATION
randomly chosen for each experiment (two-tailed unpaired Student’s t-test, ***P
< 0.001). (A-D) Co-60 was used as the source of γ radiation.
4A, middle lane). Notably, additional radiation with injection of
DHBC-MeRho caused evident fluorescence in the tumor samples
(Figure 4A, bottom lane). By randomly choosing 20 screenshots
from the microscopy of the tumor slices, the MeRho signal was
found to be statistically greater than that in the groups without
irradiation (Figure 4B). These data provide definitive proof that
radiation-induced hydroxylation of the electron-rich aromatic ring
linker is applicable for in vivo controlled release of a client
molecule. In the future, the client MeRho might be replaced by
other fluorophores with different excitation/emission wavelengths
for different experimental purposes[22]; for instance, probing
hydroxyl radicals in vivo with a DHBC-masking near-infrared
fluorogenic probe.
In fact, fluorophores are known for their high radio-sensitivity,
and most fluorophores can be easily bleached with reactive
oxygen species induced by high-energy radiation[23]. Encouraged
by the success of the radiation-induced release of fluorophores in
vitro and in vivo, we used MMAE as a model anticancer molecule
to invent a radiation-activated prodrug (RAP) by taking the
aforementioned DHBC strategy. The prodrug was denoted as
DHBC-MMAE, and its release process is shown in Figure 5A. By
treating 10 µM of DHBC-MMAE with a series doses of radiation,
we found that the release was more efficient (40 nmol/Gy, Figure
S16) than that of DHBC-MeRho (16 nmol/Gy).
To test whether the radiation-mediated cleavage reaction
could occur in living cells, we incubated 10 µM DHBC-MeRho in
4T1, HeLa and MC38 cancer cells. The cytotoxicity of DHBC-
MeRho was evaluated systematically (Figure S13), and the cell
viability of 10 µM DHBC-MeRho was greater than 95% and no
acute cytotoxicity was observed after irradiation (Figure S14). The
treated cells showed no green fluorescence, indicating no
spontaneous release of MeRho in vitro (Figure 3A, left lane).
Notably, treating DHBC-MeRho-incubated HeLa cells with γ-rays
rendered the appearance of strong green fluorescence from free
MeRho (Figure 3A, right lane). Then, the cells were irradiated with
γ-rays at different dose, and then subjected to confocal imaging.
The fluorescence intensity increased with increasing radiation
dose. After subtracting the cell autofluorescence background, the
fluorescence intensity in 4T1 cells increased by 2.6-, 6.6-, 12-,
and 18-fold under 1 Gy, 5 Gy, 10 Gy, and 20 Gy of radiation,
respectively (Figure 3B). The similar experimental results were
obtained in HeLa cells (Figure 3C) and MC38 cells (Figure 3D).
This suggests that radiation-mediated cleavage chemistry
remains functional in live mammalian cells.
A
B
***
400
300
200
100
0
D
B
C
4T1
***
***
***
80
60
40
20
0
[DHBC-MMAE]=200 nM
MMAE
100
100
80
60
40
20
0
0
Prodrug
80
0
60
0
N. S.
40
0
20
0
0
0
200 μm
0
2
4
6
8
10 12
-2
0
1
2
3
4
1
2
3
4
5
6
7
-1
Log concentration (nM)
Dose (Gy)
Fluorescence
Bright field
Merge
Figure 4. Assay of radiation-induced hydroxylation release of MeRho from the
DHBC-MeRho conjugates in tumor-bearing mice. (A) Representative confocal
fluorescence images of tumor sections. Scale bar, 200 µm (left). (B) Numbers
of MeRho-positive cells in the examined tumor sections (N=3 for both DHBC-
MeRho only and DHBC-MeRho+radiation; two-tailed unpaired Student’s t-test,
***P < 0.001). BALB/c mice implanted subcutaneously with 4T1 cells were used.
Figure 5. Radiation-triggered controlled release of MMAE—a potent anti-tumor
reagent. (A) Chemical structure of the MMAE prodrug. (B) Cell viability curve
(4T1 cells) of MMAE (IC50=1.05±0.16 nM, n=5) and DHBC-MMAE
(IC50=28.15±5.79 nM, n=5). (C) Cell viability assay of radiation-induced
controlled release of MMAE in vitro ([DHBC-MMAE]=10 nM, n=5, two-tailed
unpaired Student’s t-test, ***P < 0.001). (D) Drug released with a dose gradient
of radiation ([DHBC-MMAE]=200 nM, Co-60 as the γ radiation source, 1
Gy/min).
We then examined whether radiation could trigger
hydroxylation of DHBC-MeRho and liberate the client fluorophore
in tumor-bearing mice. In this experiment, a medical linear
accelerator (LINAC) was used as the external beam radiation
treatment for tumor-bearing mice. It delivers high-energy X-rays
to the region of the tumor. We first demonstrated that the X-ray
beam can induce the release of the client fluorescent molecule
(Figure S15), and the release efficiency was approximately 14
nmol/Gy, which is comparable to the γ-rays from Co-60 (16
nmol/Gy). Twenty microliters of 200 μM (1% DMF as a co-solvent)
DHBC-MeRho in PBS was locally injected into the 4T1 tumors,
which were treated with 4 Gy of X-rays half an hour post injection.
The treatment was repeated twice in the following two days. Then,
the tumors were harvested and snap-frozen for fluorescence
imaging[21]. When only DHBC-MeRho was injected into the mice,
no green fluorescence was detected in the tumor sections (Figure
For the in vitro experiment, 10 nM was chosen as the
evaluating concentration according to the cell viability curve of
DHBC-MMAE and free MMAE in 4T1 cells (Figure 5C) and 4 Gy
of absorbed radiation dose was chosen because 4 Gy is the
standard single-dose to treat cancer patient in clinics. As shown
in Figure 5D, viabilities of 4T1 cells without treatment (control
group, 1) and groups treated by 4 Gy radiation only (2), 10 nM of
DHBC-MMAE only (3), 10 nM of DHBC-MMAE+4 Gy radiation
(4), 10 nM of DHBC-MMAE pre-treated with 4 Gy radiation (5),
cells pre-treated with 4 Gy radiation+10 nM of DHBC-MMAE pre-
treated with 4 Gy radiation (6), and 10 nM of MMAE (7) have been
evaluated. The above experiments show that cell viabilities of
prodrug group and radiation group are >90%. As desired, cell
viabilities of prodrug+radiation group and radiation+pre-activated
This article is protected by copyright. All rights reserved.

10.1002/anie.202005612
Angewandte Chemie International Edition
COMMUNICATION
[4]
[5]
[6]
A. Gautier, C. Gauron, M. Volovitch, D. Bensimon, L. Jullien, S. Vriz, Nat.
Chem. Biol. 2014, 10, 533-541.
prodrug group dropped to ~30%, which are lower than pre-
activated prodrug group but slightly higher than free MMAE group,
indicating that radiation-induced cleavage chemistry is efficient in
vitro though not complete, which also coordinates our next finding.
According to our previous study, the local concentration of
prodrug in tumor which is delivered by antibody-drug conjugate is
about 200 nM[1c]. The prodrug molecule (200 nM) was dissolved
in PBS and irradiated with γ-rays of 1 Gy/min for a series of
periods. The concentration of released free MMAE was
determined by LC-MS, and it increased linearly with the radiation
dose, and found that >25% of MMAE (>50 nM) would be released
at the dose of 4 Gy (Figure 5D). Considering the fact that IC50 of
MMAE to many cancer cell lines is ~1 nM, the clinical application
of radiation-responsive ADC would be promising.
M. M. Lerch, M. J. Hansen, G. M. van Dam, W. Szymanski, B. L. Feringa,
Angew. Chem. Int. Ed. 2016, 55, 10978-10999.
B. K. Agrawalla, Y. Chandran, W.-H. Phue, S.-C. Lee, Y.-M. Jeong, S. Y.
D. Wan, N.-Y. Kang, Y.-T. Chang, J. Am. Chem. Soc. 2015, 137, 5355-
5362.
[7]
J. Peng, A. Samanta, X. Zeng, S. Han, L. Wang, D. Su, D. T. B. Loong,
N.-Y. Kang, S.-J. Park, A. H. All, W. Jiang, L. Yuan, X. Liu, Y.-T. Chang,
Angew. Chem. Int. Ed. 2017, 56, 4165-4169.
[8]
[9]
G. Lan, K. Ni, R. Xu, K. Lu, Z. Lin, C. Chan, W. Lin, Angew. Chem. 2017,
129, 12270-12274.
a) W. Fan, B. Yung, P. Huang, X. Chen, Chem. Rev. 2017, 117, 13566-
13638; b) K. Ni, G. Lan, S. S. Veroneau, X. Duan, Y. Song, W. Lin, Nat.
Commun. 2018, 9, 4321; c) W. P. Levin, H. Kooy, J. S. Loeffler, T. F.
DeLaney, Br. J. Cancer 2005, 93, 849-854.
Here we presented a strategy in which γ-rays and X-rays can
initiate the hydrolysis of water that produces •OH to release
fluorescent molecules and prodrugs. This strategy may also work
with other emerging radiotherapy methods, including peptide-
radionuclide radiotherapy and proton therapy, which could
produce high levels of hydroxyl radicals in tumors. Moreover,
compared with other activation methods[24], radiotherapy for
prodrug activation could have the following merits: 1) high spatial
and temporal resolution; 2) high tissue-penetration; and 3) high
clinical relevance. This combination of chemotherapy and precise
radiotherapy is likely to bring about a crucial breakthrough in
cancer treatment.
[10] R. Baskar, K. A. Lee, R. Yeo, K.-W. Yeoh, Int. J. Med. Sci. 2012, 9, 193.
[11] J.-F. Bosset, L. Collette, G. Calais, L. Mineur, P. Maingon, L. Radosevic-
Jelic, A. Daban, E. Bardet, A. Beny, J.-C. Ollier, New Engl. J. Med. 2006,
355, 1114-1123.
[12] X. Wang, X. Wang, S. Jin, N. Muhammad, Z. Guo, Chem. Rev. 2019,
119, 1138-1192.
[13] N. Kotagiri, G. P. Sudlow, W. J. Akers, S. Achilefu, Nat. Nanotechnol.
2015, 10, 370.
[14] R. K. Gill, G. S. Mitchell, S. R. Cherry, Phys. Med. Biol. 2015, 60, 4263.
[15] C. Ran, Z. Zhang, J. Hooker, A. Moore, Mol. Imag. Biol. 2012, 14, 156-
162.
[16] D. Duan, H. Liu, Y. Xu, Y. Han, M. Xu, Z. Zhang, Z. Liu, ACS Appl. Mater.
Interfaces 2018, 10, 5278-5286.
[17] P. Klán, T. Šolomek, C. G. Bochet, A. Blanc, R. Givens, M. Rubina, V.
Popik, A. Kostikov, J. Wirz, Chem. Rev. 2013, 113, 119-191.
[18] G. Albarran, E. Mendoza, R. H. Schuler, Radiat. Phys. Chem. 2016, 124,
46-51.
Acknowledgements
[19] X. Bai, Y. Huang, M. Lu, D. Yang, Angew. Chem. 2017, 129, 13053-
13057.
This work was funded by the National Natural Science Foundation
of China (no. NSFC U1867209 and no. NSFC 21778003) and the
Ministry of Science and Technology of the People’s Republic of
China (2017YFA0506300).
[20] a) H. Li, X. Li, W. Shi, Y. Xu, H. Ma, Angew. Chem. 2018, 130, 13012-
13016; b) H. Li, W. Shi, X. Li, Y. Hu, Y. Fang, H. Ma, J. Am. Chem. Soc.
2019, 141, 18301-18307.
[21] J. C. Er, C. Leong, C. L. Teoh, Q. Yuan, P. Merchant, M. Dunn, D. Sulzer,
D. Sames, A. Bhinge, D. Kim, S.-M. Kim, M.-H. Yoon, L. W. Stanton, S.
H. Je, S.-W. Yun, Y.-T. Chang, Angew. Chem. Int. Ed. 2015, 54, 2442-
2446.
Keywords: radiation-cleavable chemistry • hydroxyl radical •
fluorescent probe • prodrug activation • controlled release
[22] S. Ye, J. J. Hu, D. Yang, Angew. Chem. Int. Ed. 2018, 57, 10173-10177.
[23] R. Zhang, J. Zhao, G. Han, Z. Liu, C. Liu, C. Zhang, B. Liu, C. Jiang, R.
Liu, T. Zhao, M.-Y. Han, Z. Zhang, J. Am. Chem. Soc. 2016, 138, 3769-
3778.
[1]
a) Y. Nihongaki, F. Kawano, T. Nakajima, M. Sato, Nat. Biotechnol. 2015,
33, 755-760; b) S. T. Laughlin, J. M. Baskin, S. L. Amacher, C. R.
Bertozzi, Science 2008, 320, 664-667; c) Q. Wang, Y. Wang, J. Ding, C.
Wang, X. Zhou, W. Gao, H. Huang, F. Shao, Z. Liu, Nature 2020, 579,
421-426.
[24] a) X. Ji, Z. Pan, B. Yu, L. K. De La Cruz, Y. Zheng, B. Ke, B. Wang,
Chem. Soc. Rev. 2019, 48, 1077-1094; b) Y. Zheng, X. Ji, B. Yu, K. Ji,
D. Gallo, E. Csizmadia, M. Zhu, M. R. Choudhury, L. K. C. De La Cruz,
V. Chittavong, Z. Pan, Z. Yuan, L. E. Otterbein, B. Wang, Nat. Chem.
2018, 10, 787-794.
[2]
[3]
M. Gomelsky, Sci. Transl. Med. 2017, 9, eaan3936.
J. Wang, Y. Liu, Y. Liu, S. Zheng, X. Wang, J. Zhao, F. Yang, G. Zhang,
C. Wang, P. R. Chen, Nature 2019, 569, 509-513.
This article is protected by copyright. All rights reserved.

10.1002/anie.202005612
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
A new masking group that can be
selectively and efficiently removed
by external radiation is developed
by utilizing both the unique
aromatic hydroxylation and •OH
from the radiolysis of H2O. This
radiation-induced cleavage is
compatible with living systems, as
demonstrated by switching on an
•OH-responsive fluorescent probe
in three cancer cell lines and
mouse tumor xenografts, revealing
that external radiation could be an
efficient strategy to induce local
hydroxylation in vivo.
Q. F. Fu, H. Y. Li, D. B. Duan, C. L.
Wang, S. Y. Shen, H. M. Ma and Z.
B. Liu*
e-aq, H+, H•, •OH, H2, H2O2
H2O
2.63
0.55 2.72 0.45 0.68
•OH
Page No. – Page No.
External Radiation–Induced Local
Hydroxylation Enables Remote
Release of Functional Molecules
in Tumors
DHBO-MeRho
MeRho
γ-Ray radiation
×
non-fluorescence
green-fluorescence
This article is protected by copyright. All rights reserved.