Radical-Mediated Thiol-Ene Strategy: Photoactivation of Thiol-Containing Drugs in Cancer Cells

Article abstract of DOI:10.1002/anie.201811338

Photoactivated drugs provide an opportunity to improve efficacy alongside reducing side-effects in the treatment of severe diseases such as cancer. Described herein is a photoactivation decaging method of isobutylene-caged thiols through a UV-initiated thiol-ene reaction. The method was demonstrated with an isobutylene-caged cysteine, cyclic disulfide-peptide, and thiol-containing drug, all of which were rapidly and efficiently released under mild UV irradiation in the presence of thiol sources and a photoinitiator. Importantly, it is shown that the activity of histone deacetylase inhibitor largazole can be switched off when stapled, but selectively switched on within cancer cells when irradiated with non-phototoxic light.

Full text of DOI:10.1002/anie.201811338

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Accepted Article
Title: Radical-mediated thiol-ene strategy for photoactivation of thiol-
containing drugs in cancer cells
Authors: Shuang Sun, Bruno Oliveira, Gonzalo Jiménez-Osés, and
Gonçalo J. L. Bernardes
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201811338
Angew. Chem. 10.1002/ange.201811338
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10.1002/anie.201811338
Angewandte Chemie International Edition
COMMUNICATION
Radical-mediated thiol-ene strategy for photoactivation of thiol-
containing drugs in cancer cells
Shuang Sun,[a] Bruno L. Oliveira,[a] Gonzalo Jiménez-Osés,[b] and Gonçalo J. L. Bernardes*[a,c]
Abstract: Photo-activated drugs provide an opportunity to improve
efficacy alongside reducing side-effects in the treatment of severe
diseases, such as cancer. Herein, we describe a photoactivation
decaging method of isobutylene-caged thiols through a UV-initiated
thiol-ene reaction. The method was demonstrated with an
isobutylene-caged cysteine, cyclic disulfide-peptide and thiol-
containing drug, all of which were rapidly and efficiently released
under mild UV irradiation in the presence of thiol sources and a
photoinitiator. Importantly, we show that the activity of histone
deacetylase inhibitor Largazole can be switched-off when stapled, but
selectively switched-on when irradiated with non-phototoxic light in
cancer cells.
reactions. There are several desirable features of a click reaction,
rapid reaction rates, ease of implementation, high yields and rates
of conversion[8], so thiol-ene ‘click’ reactions have been
increasingly used for various applications, such as
biofunctionalization[9], surface and polymer modification,
polymerization[10], and so on[8, 11]. Recently, isobutylene-bridged
polymer networks have been extensively studied to synthesize
polymer networks through radical-initiated thiol-ene chemistry[12]
.
This covalently cross-linked network is able to undergo photo-
mediated, reversible cleavage of its isobutylene backbone to
allow chain rearrangement and relieve of structural strain. This
method has also been used to provide a reactive handle for
reversible addition and exchange of biochemical moieties under
cytocompatible conditions[12d]
. Key to this reaction is the
In recent decades, precision medicine has drawn a lot of attention
for the effective treatment of various diseases, especially cancer.
Currently, as a result of a lack of selectivity in the pathological
sites, the development of new, effective and safe therapies
remains challenging. Among various new methods, the recently
developed light-mediated treatment is recognized as a promising
approach to achieve controlled activation of medicine at
pathological sites[1], which could significantly reduce the side
effects of chemotherapy. So far, in the battle against cancer,
several types of light-activated anti-cancer reagents, which can
be switched on conditionally with irradiation, have been
investigated[2]. The structures of these photocaged drugs include
various ultraviolet, near infra-red or visible responsive moieties[3],
such as o-nitrobenzyl[2a, 4], coumarinyl ester[5], metal complexes[2e,
6]. These photo-responsive structures offer an extensive toolbox
for use in cancer therapies and other biological applications.
However, issues and challenges remain in this field, such as using
non-toxic wavelength, achieving rapid and efficient conversion
and improving the bioavailability of the prodrug. Therefore, there
is still demand for new designs and new developments for photo-
mediated therapy.
isobutylene structure capable of addition-fragmentation chain
transfer (AFCT), in which the structure is attacked by the photo-
initiated thiol radical in the presence of a photoinitiator (PI) to
release the caged thiol part.
Scheme 1. a. General thiol-ene coupling reaction; b. the thiol-isobutylene
decaging reaction.
Inspired by this AFCT reaction, we hypothesized that the
isobutylene structure could be used as a bridging graft to cage
thiol-drugs and allow further controlled activation of anti-cancer
drugs by means of a radical-mediated thiol-ene mechanism
(Scheme 1). Previously, we reported a one-pot macrocyclization
strategy [with tris(2-carboxyethyl)phosphine (TCEP)] for thiol-
containing peptides by using an isobutylene graft, which can
significantly enhance both membrane permeability and binding
activity of the corresponding macrocycles[13]. The isobutylene
graft can be rapidly and efficiently installed onto reduced thiol
groups in a biocompatible manner because of the high reactivity
of the bis-bromo isobutylene. Our research began with N-tert-
butyloxycarbonyl(Boc)-L-cysteine 1, which was protected with bis-
bromo isobutylene (Table 1 and Supporting Information). Stapled
cysteine 2 was then screened under a series of conditions. Firstly,
different PIs were tested with the same amounts of thiol source,
1-thio-β-D-glucose tetraacetate, and stapled cysteine under
irradiation at 365 nm. The reaction with water-soluble PI 2,2’-
azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (Vazo 44)
did not progress after 2 hours. However, the other PI, 2,2-
Thiol-ene reactions (Scheme 1), a conjugation between a thiol
and an alkene, have been known since the early 1900s[7]. The
coupling reaction proceeds through two mechanisms, photo-
initiated free-radical addition and catalyzed Michael addition
[a]
S. Sun, Dr. B. L. Oliveira, Dr. G.J.L. Bernardes
Department of Chemistry
University of Cambridge
Lensfield Road, CB2 1EW Cambridge (UK)
E-mail: gb453@cam.ac.uk
[b]
[c]
Dr. G. Jiménez-Osés
Departamento de Química. Centro de Investigación en Síntesis
Química. Universidad de La Rioja. 26006 Logroño (Spain)
Dr. G.J.L. Bernardes
Instituto de Medicina Molecular
Faculdade de Medicina, Universidade de Lisboa
Avenida Professor Egas Moniz, 1649–028 Lisboa (Portugal)
E-mail: gbernardes@medicina.ulisboa.pt
Supporting information for this article is given via a link at the end of
the document.
dimethoxy-2-phenylacetophenone
promoted the reaction and released N-Boc-cysteine 1. β-
(DPAP)
successfully
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Mercaptoethanol (BME) was also tested as the thiol compound
under the same conditions. This reaction released the cysteine in
a slightly lower yield, which suggests that different thiol sources
could be used to promote the reaction. However, when N,N-
dimethylformamide (DMF) was used as solvent, two disulfides
between the thiol-glucose and N-Boc-cysteine 1, and N-Boc-
cystine were observed. To evaluate the reaction and calculate the
isolated yield, TCEP was added to reduce the disulfides after the
reaction. Under these conditions, the reaction was complete
within 15 mins and gave a relatively high yield (65%). Moreover,
the reaction was also rapid and efficient when glutathione was
used as a thiol source in a mixed solvent of DMF and water, which
shows that the reaction is practical for an aqueous environment.
Then, the feasibility of our strategy was investigated with an
isobutylene cyclized 5-mer peptide that bears two terminal
Figure 1. Thiol-ene decaging reaction of isobutylene-cyclized peptide. a. The
decaging reaction of stapled CAAAC peptide with 1-thio-β-D-glucose
tetraacetate. b. HPLC traces of the starting material and reaction mixture. Blue,
stapled CAAAC peptide; Red, reaction mixture after 15 min.
cysteines and synthesized by solid-phase peptide synthesis[13]
.
The short peptide was completely converted into the disulfide
derivative within 15 mins under the same conditions described
previously (Figure 1).
The proposed mechanism for the radical-mediated thiol-ene
decaging reaction was studied by Quantum Mechanical
calculations using abbreviated thiol models (see Supporting
Information) and is shown as Scheme 2. After generation of the
thiol radical by the PI under UV irradiation, the isobutylene grafted
structure undergoes a fast thiol-ene anti-Markovnikov addition
with a calculated activation energy of ΔG‡  14 kcal mol–1 at the
PCM(H2O)/M06-2X/6-31++G(2,p) theory level, to generate a
symmetric tertiary carbon-centered radical intermediate. Then,
the unstable radical intermediate undergoes a β-scission at a very
similar reaction rate regenerating the isobutylene linkage and
resulting in a mixed caged compound, which can be again
attacked by another thiol radical following the same mechanism
to release the other unit of the caged thiol compound. The process
is nearly thermoneutral and reversible until two decaged radical
thiols collapse forming a stable disulfide bond (Scheme 2).
Table 1. Optimization of the thiol-ene decaging reaction.
Time
Yield
Entry
PI
Thiol
Solvent
TCEP
(min)
(%)
1
2
3
4
5
6
Vazo44
DPAP
DPAP
DPAP
DPAP
DPAP
4AcGlcSH
4AcGlcSH
BME
MeOH
CH2Cl2
CH2Cl2
DMF
-
120
120
120
120
15
0
-
54
-
37
[a]
4AcGlcSH
4AcGlcSH
4AcGlcSH
-
Table 2. Control studies of thiol-ene decaging reactions between
isobutylene-grafted cysteine and thiol-glucose.
[a]
DMF
-
DMF
+
15
65
Stapled
Cys 2
Conversion
(%)
Entry
Thiol
UV
DPAP
TEMPO
DMF/H2O
1/1
7
DPAP
GSH
+
15
67
1
2
+
+
+
-
+
+
-
+
-
+
+
+
+
+
-
-
100
[a]A mixture of disulfides between cysteine and thiol-glucose and cystine. PI,
Photoinitiator; 4AcGlcSH, 1-thio-β-D-glucose tetraacetate; BME, β-
mercaptoethanol; GSH, glutathione.
no reaction
no reaction
no reaction
no reaction
3
+
+
+
-
To demonstrate that the release of the thiol compound is by
means of a radical-mediated thiol-ene mechanism, a series of
control experiments were conducted (Table 2 and Supporting
Information). As shown in the table, a thiol source, UV irradiation
and a PI are essential for the decaging reaction. When radical
scavenger (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) was
added to the reaction, it terminated the reaction by forming an
intermediate with the DPAP fragmentation, which suggests that
the reaction is mediated by radicals.
4
+
+
-
5[a]
+
+
[a]A mixture of disulfides between cysteine and thiol-glucose and cystine. PI,
Photoinitiator; 4AcGlcSH, 1-thio-β-D-glucose tetraacetate; BME, β-
mercaptoethanol; GSH, glutathione.
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10.1002/anie.201811338
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Scheme 2. Proposed mechanism of the radical-mediated thiol-isobutylene
decaging reaction.
With all this knowledge in hand and to demonstrate that this
strategy is practical to activate drugs in vitro, we applied our
method to potent histone deacetylase inhibitor (HDAC) Largazole.
Cyclic depsipeptide Largazole is a marine natural product, and its
derivatives are recognized as promising potential anticancer
therapeutics. Cyclic depsipeptide Largazole is a marine natural
product, and its derivatives are recognized as promising potential
anticancer therapeutics. Largazole possesses remarkable and
preferential growth-inhibitory activity against cancer cell lines
relative to corresponding non-transformed cells[14]. The octanoyl
tail in Largazole has better cell-permeability than the active free
thiol species, Largazole thiol, the latter of which is formed inside
cells by esterase or lipase-based cleavage of the octanoyl
residue[15]. The free thiol group binds to the active site Zn2+-
domain within the HDAC enzyme, which results in a potent
inhibitory effect. Thus, the thiol-ene decaging strategy can be
used to protect the thiol group, improve the cell-permeability and
allow controlled activation with UV. Therefore, we synthesized
three Largazole derivatives; Largazole, Largazole thiol and
stapled Largazole (see Supporting Information). The result of the
parallel artificial membrane permeability assay indicated that
stapled Largazole is a highly passively permeable compound
(logPe –5.29 and Pe 5.3x10–6 cm/sec; Supporting Information
Table S4). Then, the stapled Largazole was reacted with 1-thio-
β-D-glucose tetraacetate and DPAP under UV irradiation for 15
min (Figure 2a). Full conversion of the stapled Largazole was
observed in the HPLC trace along with the appearance of
Largazole thiol signal (Figure 2b). Next, the growth-inhibitory
activity was evaluated with human colon carcinoma cell lines,
HCT-116 (Figure 2c). As expected[16], Largazole (GI50 1.433 nM)
is more potent than the corresponding free thiol species (GI50
185.1 nM) owing to its octanoyl side-chain which improves cell
permeability and allows facile presentation of the free thiol within
the cell[15]. The stapled Largazole (GI50 407.7 nM) is a much less
potent compound because the thiol group is protected by the
isobutylene structure, which prevents binding with the Zn2+
domain in cells. Before testing the decaging condition in cells, we
investigated the toxicity of DPAP and phototoxicity of the light in
terms of the power and the irradiation time (see Supporting
Information, Figure S7). A set of cytocompatible conditions, 15
min irradiation at 80 W, were chosen to conduct further
investigations.
Figure 2. The photoactivation of isobutylene-grafted Largazole thiol. a. Thiol-
ene decaging reaction of stapled Largazole with 1-thio-β-D-glucose tetraacetate.
HPLC traces of the reaction mixture. Blue, stapled Largazole; Red, reaction
mixture after 15 min. b. Growth inhibitory effects of Largazole, Largazole thiol
and stapled Largazole on HCT-116 colon carcinoma cells, Largazole GI50 1.433
nm, Largazole thiol GI50 185.1 nm, stapled Largazole GI50 407.7 nm. c. Cell
survival under different conditions; SL, stapled Largazole, UV condition, 365 nm
80 W, 15 min. d. HDAC activity assay of control group and UV group. Data are
representative of three independent tests and analyzed by the two-tailed
unpaired Student’s t-test. **, P < 0.01; ****, P < 0.0001. Error bars reflect one
standard deviation from the mean.
The decaging reaction of stapled was tested with HCT-116
cells at 150 nM. McCoy's 5A culturing medium contains cysteine
and glutathione, so no other thiol source was added to the
medium. The cell viabilities of the three drug groups with/without
UV irradiation were consistent with the growth-inhibitory assay
(Figure 2d). The UV-irradiated premixed group of stapled
Largazole and DPAP showed significantly lower cell viability than
the corresponding non-irradiation group, the stapled Largazole
group and the Largazole thiol group. To confirm the results, a
fluorometric HDAC activity assay was conducted with HCT-116
cell lysates (Figure 2e). A significant decrease of fluorescence
was observed in the UV-irradiated premixed group relative to the
control groups and the non-irradiation group. Both the cell viability
and the enzyme activity results indicated that the stapled
Largazole was successfully activated by UV light.
In summary, we have developed a rapid and efficient thiol-ene
based photoactivation strategy for thiol-containing drugs, caged
with isobutylene. The radical-mediated reaction, that is triggered
by UV light, undergoes a thiol-ene addition step to form an
unstable radical intermediate, which is further cleaved by β-
scission to release the caged thiols. We applied this method to
various substrates, such as N-Boc-cysteine,
a cysteine-
containing peptide and the HDAC inhibitor Largazole, and
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Acknowledgements
Funded under the Royal Society (URF to G.J.L.B.), FCT Portugal
(iFCT to G.J.L.B.), ERC StG (grant agreement No. 676832), D.G.I.
MINECO/FEDER (CTQ2015-70524-R and RYC-2013-14706 to
G.J.O.), Cambridge Trust and China Scholarship Council (PhD
studentship to S.S.) and the EPSRC. The authors thank Vikki
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Keywords: photoactivation • caged drugs • radical reaction •
thiol-ene • isobutylene
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10.1002/anie.201811338
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COMMUNICATION
COMMUNICATION
An UV-initiated thiol-ene
reaction enables
Shuang Sun, Bruno L. Oliveira,
Gonzalo Jiménez-Osés and Gonçalo
J. L. Bernardes*
photoactivation-mediated
decaging of isobutylene-caged
thiols. With this strategy, the
activity of histone deacetylase
inhibitor Largazole can be
switched-off when stapled, but
selectively switched-on when
irradiated with non-phototoxic
light in cancer cells.
Page No. – Page No.
Radical-mediated thiol-ene strategy
for photoactivation of thiol-
containing drugs in cancer cells
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