Photoswitchable anticancer activity via trans-cis isomerization of a combretastatin A-4 analog

Article abstract of DOI:10.1039/c5ob02005k

Combretastatin A-4 (CA4) is highly potent anticancer drug that acts as an inhibitor of tubulin polymerization. The core of the CA4 structure contains a cis-stilbene, and it is known that the trans isomer is significantly less potent. We prepared an azoben

Full text of DOI:10.1039/c5ob02005k

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Photoswitchable anticancer activity via trans–cis
isomerization of a combretastatin A-4 analog†
Cite this: DOI: 10.1039/c5ob02005k
Jonathon E. Sheldon,a,d M. Michael Dcona,b,d Charles E. Lyons,d John C. Hackettc,d
Received 25th September 2015,
Accepted 19th October 2015
and Matthew C. T. Hartman*a,d
DOI: 10.1039/c5ob02005k
www.rsc.org/obc
Combretastatin A-4 (CA4) is highly potent anticancer drug that ery are promising, in all cases the drug activation is irreversible
acts as an inhibitor of tubulin polymerization. The core of the CA4 upon illumination. Very recently, Presa and coworkers have
structure contains a cis-stilbene, and it is known that the trans developed Pt(II) complexes with ligands that can be switched
isomer is significantly less potent. We prepared an azobenzene between more toxic and less toxic forms depending on the
analog of CA4 (Azo-CA4) that shows 13–35 fold enhancement in wavelength of light used.14 One potential problem for the clini-
potency upon illumination. EC50 values in the light were in the mid cal application of each of these light-activated drug systems is
nM range. Due to its ability to thermally revert to less toxic trans that the activated drug can diffuse away from the illuminated
form, Azo-CA4 also has the ability to automatically turn its activity site of the tumor, causing unwanted damage to the tissues it
off with time. Azo-CA4 is less potent than CA-4 because it encounters. An ideal light-activated drug would automatically
degrades in the presence of glutathione as evidenced by UV-Vis revert to a less potent form over time in order to limit this off-
spectroscopy and ESI-MS. Nevertheless, Azo-CA4 represents a target toxicity. Photoswitchable drugs also offer other unique
promising strategy for switchable potency for treatment of cancer.
opportunities in photopharmacology.15
CA4 (Fig. 1) is a tubulin polymerization inhibitor and anti-
cancer drug that exhibits selective cytotoxicity toward tumor
vasculature.16,17 A variety of cancer cells are highly sensitive to
CA4 treatment. EC50 values are often under 10 nM.18,19
A prodrug of CA4 (CA4-phosphate) is currently being tested in
clinical trials in combination with existing chemotherapy
agents.20,21 In addition, a photocaged version of CA4 has been
developed to monitor microtubule dynamics in cells.22 CA4
contains a cis-stilbene structure (Fig. 1a), which is known to be
60-fold more potent than the trans isomer.23 Light could be
used to switch between the two isomers, but this process
requires <300 nm UV light which is toxic to cells.24 Azoben-
zenes contain a molecular backbone that resembles the stilbe-
noid backbone of CA4, with the olefinic carbon atoms replaced
with nitrogens (Fig. 1a). Yet, unlike stilbenes, azobenzenes are
able to be switched from the trans to cis form with low inten-
sity light that is compatible with cells.25,26 Moreover, when
irradiation is terminated, the cis form of azobenzenes relaxes
back to the thermodynamically stable trans form with a half-
life that ranges from milliseconds to hours.27–29 In principle,
therefore, an azobenzene analog of CA4 (Azo-CA4, Fig. 1a) will
exhibit the dual properties we desired: enhancement in
potency with light and automatic turn-off over time.
Introduction
Off-target toxicity persists as a challenge in the development
and improvement of cancer chemotherapeutics. The use of
light is one means to target therapy at the tumor site. In photo-
dynamic therapy (PDT), a photosensitizer is employed to
create cytotoxic reactive oxygen species (ROS) at a point of
irradiation.1,2 One of the clinical challenges involving PDT is
that hypoxic tumor microenvironments can limit the ability to
create sufficient ROS for therapy.3,4 As an alternative, research-
ers have used light to trigger drug activation or drug delivery.
Examples include the release of anti-cancer drugs from a nega-
tively charged, cell-impermeable small molecule along with
release from cancer-targeting dendrimers, nanoparticles and
liposomes.5–13 Although these methods for targeted drug deliv-
aDepartment of Chemistry, Virginia Commonwealth University, 1001 W. Main St.,
Richmond, VA 23284-2006, USA. E-mail: mchartman@vcu.edu
bDepartment of Internal Medicine, Virginia Commonwealth University,
1101 E. Marshall St., Richmond, VA 23298-0663, USA
cDepartment of Physiology and Biophysics, Virginia Commonwealth University,
1101 E. Marshall St., Richmond, VA 23298-0551, USA
dMassey Cancer Center, Virginia Commonwealth University, 401 College St.,
Very recently, Borowiak et al.30 and Engdahl et al.31 reported
the synthesis of azobenzene analogs of CA-4 and have extensively
investigated their use as tools for controlling tubulin polymeriz-
ation in cells. They have also reported EC50 values using various
Richmond, VA 23292-0037, USA
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob02005k
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lamp used in all studies consisted of a simple aquarium light
fixture containing two Philips PL-S 9W/2P BLB bulbs. The light
was placed at distance of 5 cm from the top of plate. The inten-
sity was measured to be 4.4 mW cm−2
.
Compound synthesis
3-((tert-Butyldimethylsilyl)oxy)-4-methoxynitrobenzene,
1.
To a solution of 2-methoxy-5-nitrophenol (5 g, 29.6 mmol) in
DMF (120 mL), TBDMS-Cl (4.90 g, 32.5 mmol) was added in
one portion. The reaction mixture was stirred vigorously for
3 hours until TLC showed disappearance of 2-methoxy-5-nitro-
phenol. The mixture was evaporated and water (80 mL) was
added. This solution was extracted 3 times with CH2Cl2
(80 mL) and the combined organic layers were washed with
brine, dried over Na2SO4, and evaporated to yield 7.73 g
(92.3%) of a brown solid. 1H NMR (400 MHz, CDCl3) δ 7.89
(dd, J = 9.0, 2.8 Hz, 1H), 7.71 (d, J = 2.7 Hz, 1H), 6.89 (d, J =
9.0 Hz, 1H), 3.92 (s, 3H), 1.01 (s, 9H), 0.19 (s, 6H). 13C NMR
(101 MHz, CDCl3) δ 156.76, 145.01, 141.42, 118.40, 116.06,
110.48, 55.87, 25.60, 18.44, 17.39, −4.63, −4.92. ESI-MS
(M + H)+ C13H22NO4Si+ calc. 284.1313. obsd 284.1314.
3-((tert-Butyldimethylsilyl)oxy)-4-methoxyaniline, 2. Com-
pound 1 (2 g, 7.1 mmol) was dissolved in an anhydrous
1 : 1 mixture of MeOH and EtOH (24 mL) in the presence of
10% Pd/C (400 mg, 0.38 mmol) under Ar. The reaction vessel
was purged for 20 minutes with H2 gas, sealed, and stirred for
3 h. The reaction was filtered through Celite and evaporated to
1
yield 1.69 g (94.5%) of a dark brown oil. H NMR (400 MHz,
CDCl3) δ 6.68 (d, J = 8.4 Hz, 1H), 6.28 (d, J = 2.8 Hz, 1H), 6.25
(dd, J = 8.4, 2.7 Hz, 1H), 3.72 (s, 3H), 3.30 (s, 2H), 0.99 (s, 9H),
0.15 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 146.12, 144.32,
140.65, 114.43, 109.49, 108.20, 56.62, 25.76, 18.43, −4.36,
−4.64. ESI-MS (M + H)+ C13H24NO2Si+ calc. 254.1571. obsd
254.1566.
Fig. 1 (a) Design of Azo-CA4, a photoswitchable analog of CA4. (b)
RI-MP2/def2-TZVP derived electronic structure of CA4 (left) and Azo-
CA4 (right). Labeled charges for non-hydrogen atoms were computed
using the natural population analysis method.
(E)-1-(3-((tert-Butyldimethylsilyl)oxy)-4-methoxyphenol)-2-
(3,4,5-trimethoxyphenyl)diazene, 3. Compound
2
(1 g,
3.9 mmol) and 3,4,5-trimethoxyaniline (723 mg, 3.9 mmol)
were stirred in freshly distilled CH2Cl2 under Ar. (Diacetoxy-
iodo)benzene (2.54 g, 7.9 mmol) was added in one portion.
The reaction was allowed to proceed until the disappearance
of compound 2 via TLC (20 minutes). The reaction mixture
was purified by flash column chromatography (100% Hexanes
to 20% EtOAc in Hexanes) to yield 107 mg (6.3%) of an orange
solid. 1H NMR (400 MHz, CDCl3) δ 7.58 (dd, J = 8.6, 2.4 Hz,
1H), 7.45 (d, J = 2.4 Hz, 1H), 7.21 (s, 2H), 6.96 (d, J = 8.6 Hz,
light regimes. Herein we add to the knowledge of these exciting
compounds, focusing on the effectiveness of their ability to auto-
matically turn off their activity over time, their effects on endo-
thelial cells, and potential limitations for in vivo use.
Experimental
General methods
All reagents and solvents were purchased from Sigma-Aldrich, 1H), 3.97 (s, 6H), 3.92 (s, 3H), 3.90 (s, 3H), 1.03 (s, 9H), 0.20 (s,
VWR, or Fisher Scientific. Reagents for cellular assays were 6H). 13C NMR (101 MHz, CDCl3) δ 153.82, 153.51, 148.69,
purchased from ATCC, Invitrogen-Gibco, or VWR. The tubulin 146.99, 145.53, 140.16, 119.41, 113.35, 111.20, 100.16, 61.02,
polymerization assay kit was purchased from Cytoskeleton Inc. 56.22, 55.59, 29.71, 25.74, 18.50, 1.03, −4.56. ESI-MS (M + H)+
1H NMR spectra and 13C spectra were recorded at room temp- C22H33N2O5Si+ calc. 433.2153. obsd 433.2149.
erature on a Bruker 400 MHz Ultrashield Plus. Proton and
Carbon chemical shifts are expressed in parts per million (Azo-CA4) 4. To
(E)-2-Methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenol,
solution of compound (53.5 mg,
a
3
(ppm, δ scale) and are referenced to the solvent peak. UV-Vis 0.12 mmol) in DMF (4 mL), potassium fluoride hydrate
experiments were carried out on either NanoDrop ND-1000 or (81.6 mg, 1.4 mmol) and AcOH (20 μL, 0.35 mmol) were
an Agilent 8453 UV/Vis. Cell Titer Blue (CTB) fluorescence was added. The reaction was complete after 1 hour as monitored
measured on BioTek Synergy H1 Hybrid plate reader. The UV via TLC. The reaction mixture was evaporated to yield an
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orange powder. The powder was dissolved in 5 mL CH2Cl2 and Characterization of glutathione reduction products by ESI-MS
washed with H2O (3 × 5 mL), once with brine, and dried over
Samples were loaded on a self-packed fused silica (Polymicro
Na2SO4. The organic layer was purified by flash column
Technologies) trap column (360 micron o.d. × 100 micron i.d.)
chromatography (100% Hexanes to 20% EtOAc in Hexanes) to
with a Kasil frit packed with 5–15 micron irregular phenyl C-18
YMC packing. The trap column was connected to an analytical
column (360 micron o.d. × 50 micron i.d.) with a fritted tip at
5 micron or less (New Objective) packed with 5 μm phenyl C-18
YMC packing. Peptides were trapped and then eluted into a
Thermo Finnigan LCQ deca XP max mass spectrometer with
an acetonitrile gradient from 0% to 80% over one hour at a
yield 20.6 mg (53.9%) of a waxy orange solid. 1H NMR
(400 MHz, CDCl3) δ 7.56–7.51 (m, 2H), 7.22 (s, 2H), 6.98 (d, J =
8.3 Hz, 1H), 5.72 (s, 1H), 3.98 (s, 3H), 3.96 (s, 6H), 3.93 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 153.53, 149.20, 148.56, 147.44,
146.23, 140.32, 118.92, 110.09, 106.16, 100.26, 61.02, 56.20.
ESI-MS (M + H)+ C16H19N2O5 calc. 319.1288. obsd 319.1299.
flow rate of between 50–150 nl per minute. The mass spectro-
Molar extinction coefficient determination
meter was operated in data dependent mode. First a MS scan
A 3.1 mM stock solution of Azo-CA4 was prepared in 23%
DMSO in water. The solution was stirred vigorously and kept
in the dark. 400 μL of the solution was placed in the each of
three tubes and diluted to 1 mL with water to give a final con-
centration of 1.26 mM Azo-CA4. 2-fold serial dilutions were
made and the absorption of each solution was measured on a
NanoDrop ND-1000 spectrophotometer. The molar absorptiv-
ity, based on three independent trials, was found to be 19 150
from mass 200 or 300–2000 m/z was collected to determine the
mass of molecules eluting at that time, the top two most abun-
dant masses were fragmented into MS/MS scans and placed
on an exclusion list. This sequence MS followed by 2 MS/MS
scans with exclusion was repeated throughout the 15 minute
gradient. Ion chromatograms were plotted of the masses of
interest.
L mol−1 cm−1
.
In vitro tubulin polymerization
Fluorescence in vitro tubulin polymerization assays using
porcine tubulin were conducted using a fluorescence based
polymerization assay kit (Cat. #BK011P; Cytoskeleton, Denver,
CO) and following the manufacturers protocol. Each assay was
exposed to varying concentrations of Azo-CA4 (80 μM–800 nM).
Samples were either exposed to light (380 nm, irradiated for
1 minute) or kept in the dark. Irradiated samples of Azo-CA4
were added to tubulin containing wells directly after
exposure to light. Azo-CA4 samples kept in the dark were
added at the same time and the kinetic assay was
started immediately and proceeded for 1 hour at 37 °C (Ex.
350 nm, Em. 435 nm). Samples were normalized to assays
lacking Azo-CA4. IC50 values were calculated by fitting the
points to the four parameter logistic equation using SigmaPlot
software.
Half-life of thermal relaxation determination
A solution of 300 μM Azo-CA4 (100 μL) in either 10% CH3CN
in PBS, 20% CH3CN, 0.25% DMSO in PBS, 0.9% DMSO in PBS,
or 0.25% DMSO in H2O were placed in a 96 well plate and were
irradiated with 380 nm light (4.4 mW cm−2) for 1 minute.
Absorbance readings were taken from triplicate wells every
1 min for 3 h at 37 °C and subtracted from the fully relaxed
absorbance reading. Data was fitted to a 3-parameter exponen-
tial curve (SigmaPlot software).
Azo-CA4 photoswitching integrity assay
A 64 μM solution of Azo-CA4 in DMSO was placed in a cuvette
in an Agilent 8453 UV/VIS at 37 °C for 23 hours. The sample
was illuminated for 1 min every 3 hours and the absorbance
was measured every 5 minutes.
Cell lines and culture conditions
pH sensitivity experiments
The HUVEC line was cultured in the recommended Basal
media supplemented with VEGF growth factors, 5% Fetal
Bovine Serum (FBS, Life Technologies), 2% L-glutamine and
1% of a 10 000 units per mL penicillin and 10 000 μg mL−1
streptomycin solution. The MDA-MB-231 line was cultured in
DMEM media with supplemented with 10% FBS and 1% of a
10 000 units per mL penicillin and 10 000 μg mL−1 strepto-
mycin solution. The T24 line was cultured in McCoy’s 5a
Medium (ATCC) and supplemented with 10% FBS and 1% of a
10 000 units per mL penicillin and 10 000 μg mL−1 strepto-
mycin solution.
A 183 μM solution of Azo-CA4 in 3.2% DMSO in 50 mM phos-
phate buffer (pH 8.0) was prepared. The absorption spectrum
and pH were measured after multiple additions of small
volumes of concentrated HCl.
Glutathione reduction assay
Azo-CA4 (300 μM in 5.7% DMSO/PBS pH 7.2) in the presence
of fully reduced glutathione (10 mM in PBS pH 7.2) and TCEP
(5 mM in PBS pH 7.2) was placed in 3 separate wells (100 μL
per well) of a 96 well plate at 37 °C. Experiments were per-
formed under UV (380 nm) irradiation (1 min every 2 hours,
4.4 mW cm−2) or protected from light exposure. For experi-
ments in the light, absorbance readings were collected
Cell viability assays
immediately prior to the subsequent light exposure. Absor- Cells (18 750 cells per mL for HUVEC, 125 000 cells per mL for
bance readings were measured on a BioTek Synergy H1 Hybrid MDA-MB-231 and T24 125 000 cells per mL) were seeded in
plate reader.
dark, flat bottom 96 well plates with clear lids for >2 hours in
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80 μL media. After the growing period, Azo-CA4 and CA4 were lowed by reduction of the nitro group to give 2. Compound 2
added at varying concentrations in 20 μL 0.25% DMSO in was coupled to 3,4,5-trimethoxyaniline with diacetoxyiodo-
media. Various concentrations of Azo-CA4 (30 μM–58.6 nM for benzene34 to yield azobenzene 3 which was separable from
experiments in the light and 100 μM–195 nM for experiments homo-aniline coupling side products. Finally, 3 was de-
in the dark) and CA4 (30 nM–0.059 nM) were replicated 5 protected with fluoride ion to yield Azo-CA4, 4.
times on each plate. Cells were incubated with drug for
We analyzed the photoswitching properties of Azo-CA4
48 hours with one 96 well plate undergoing irradiation at (Fig. 2). The absorbance spectrum of trans azo-CA4 showed a
380 nm for 1 minute every hour or every 2 hours for the λmax of 379 nm and an extinction coefficient consistent with
duration of the experiment and the other kept in the absence other reports (Fig. 2a and ESI Fig. S1†).30,35 After 30 seconds of
of light. 5 wells in each experiment were devoted to media with irradiation with 380 nm light, the compound’s absorption
no cells or cells with vehicle. At the end of the 48 hour time pattern had dramatically shifted toward an absorption profile
period cells were incubated for 2 hours with 20 μL Cell Titer that is characteristic for cis azobenzenes36,37 (Fig. 2a). Over
Blue reagent (CTB) (Promega) at which time fluorescence was time Azo-CA4 reverted back to the trans form with a half-life of
measured on a BioTek Synergy H1 Hybrid plate reader (Ex. 88 min at 37 °C (Fig. 2b, c and ESI Fig. S2 and Table S1†). Mul-
560 nm, Em. 590 nm). Cell viability was expressed as a percen- tiple cycles of switching and relaxation could be performed
tage of maximum (cells with vehicle) and corrected for back- over 24 h without degradation (Fig. 2d). Surprisingly, the half-
ground fluorescence of the media. Data is shown as an average life for thermal reversion measured under our conditions was
S.D. of three independent experiments. GI50 values were considerably longer than that reported previously.30 To check
calculated using SigmaPlot software using a four parameter if the presence of DMSO or buffers caused this change, we
logistic equation.
measured the photoswitching kinetics under a variety of buffer
conditions and light intensities, including those identical to
that reported by Borowiak et al.,30 (Fig. S2 and S3†). Our half-
lives for reversion (75–100 min) are consistently much longer
than the reported value (6.2 min) even when measured under
identical conditions (20% CH3CN in PBS) (Table S1†).
We then demonstrated that Azo-CA4 inhibits tubulin
polymerization in a similar manner to CA4 (ESI Fig. S4†).
In vitro experiments were carried out with Azo-CA4
pre-activated with light or in the dark. As expected, in the light
Azo-CA4 was a more potent inhibitor of tubulin polymerization
than in the dark; light activated Azo-CA4 had an IC50 of
5.1 μM (ESI Fig. S4†), similar to the IC50 of CA4 (1.9 μM)38
and was 2.8-fold more potent than the dark adapted
compound.
Results
Prior to synthesis of Azo-CA4, we performed a computational
study (Fig. 1b) to compare its structure to CA4. Both molecules
were fully optimized at the RI-MP2/def2-TZVP level of theory
and natural population analyses were performed.32 Azo-CA4
was, indeed, highly similar in structure to CA4 (Fig. 1b). The
optimized geometries and natural charges calculated for non-
hydrogen atoms are almost identical with the exception of the
nitrogens on the azo bridge. Because the aromatic groups and
their methoxy substituents are the major contributors to
tubulin binding in CA4,33 based on structure alone, we would
expect that Azo-CA4 would be a potent tubulin inhibitor and
act similarly to CA4.
Borowiak et al.30 and Engdahl et al.31 investigated the toxi-
city of Azo-CA4 on several cancer cell lines. CA4 is a known
vasculature-disrupting agent, and we therefore tested the
effects of Azo-CA4 on human umbilical vein endothelial cells
(HUVECs).39,40 For comparison, we also evaluated the effects
of Azo-CA4 on a breast adenocarcinoma epithelial cell line
(MDA-MB-231) that has shown sensitivity to CA4.41 Assays with
CA4 itself in these cell lines showed EC50s in the low nM
range, consistent with other reports (ESI Fig. S5†). With Azo-
CA4, we observed significant enhancement of potency in the
presence of light vs. untreated controls for both cell lines
(Fig. 3a and b). Based on the half-lives we determined for cis
Azo-CA4, we designed several pulse patterns to check for light-
enabled toxicity ranging from a single 1 minute pulse at the
beginning of the experiment, to a 1 minute pulse every
60 minutes (Fig. 3c). As expected with the turn-off mechanism,
we saw decreasing EC50 values as the duration between pulses
decreased (Table 1). For the 1 h pulse regime, Azo-CA4 was 22-
fold more toxic to HUVEC cells than dark controls. Light alone
at the same doses was not toxic for either cell line (Fig. 3d).
The significant gap between the cytotoxicity of CA4 and
Azo-CA4 in these cell lines suggested that Azo-CA4 may be
To synthesize Azo-CA4 (Scheme 1), we first protected the
phenolic oxygen of nitroaniline 1 with a TBDMS group fol-
Scheme 1 Synthesis of Azo-CA4. Reaction conditions: (a) TBDMS-Cl,
DMF 92%. (b) 10% Pd/C, H2, MeOH/EtOH, 95%. (c) 3,4,5-Trimethoxy-
aniline, (diacetoxyiodo)benzene, CH2Cl2, 6%. (d) KF, AcOH, 95%.
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Fig. 2 Absorption experiments performed on Azo-CA4. (a) Absorption spectra of Azo-CA4 (157 μM) after exposure to 380 nm light (4.4 mW cm−2
)
for various amounts of time. (b) Absorption spectra demonstrating thermal relaxation over time of Azo-CA4 in the dark at 37 °C. Prior to placing in
the dark, Azo-CA4 was treated for 1 minute with 380 nm light (4.4 mW cm−2). (c) Time course of Azo-CA4 relaxation in DMSO monitored by absor-
bance at 380 nm. (d) Time course of 380 nm absorbance of Azo-CA4 over time in DMSO. The sample was irradiated with 380 nm light (4.4 mW
cm−2) for 1 minute every 3 hours at 37 °C (arrows). The final point indicates a fully dark-adapted Azo-CA4 left in the dark for 100 hours after the last
irradiation.
unstable in cells. Since azobenzenes are known to react with tion. The MS-MS fragmentation of this peak showed neutral
glutathione (GSH),42,43 we evaluated the extent to which GSH loss peaks of 307, 275, and 129, all of which are characteristic
could inactivate Azo-CA4 (Fig. 4 and ESI Fig. S6†). Both forms for GSH adducts (ESI Fig. S7†).44 This peak was not present in
of Azo-CA4 were degraded by GSH over the course of several a control reaction lacking Azo-CA4 (ESI Fig. S9†), lending
hours. Interestingly, the light-activated cis-Azo-CA4 compounds credence to the fact that this species is an adduct of gluta-
were degraded more rapidly than trans-Azo-CA4, and this effect thione and Azo-CA4.
could limit the potency at higher light doses when more cis-
Azo-CA4 is present.
To investigate this further, we treated our compound with
light and GSH and followed the course of the reaction by
Discussion
LC-MS/MS (Fig. 5 and ESI Fig. S7–S9†). As expected, the peak
corresponding to the cis-Azo-CA4 decreased over time (Fig. 5a
and ESI Fig. S7†). Surprisingly, a new peak of the same mass
was formed during the reaction. This peak did not co-elute
with trans-Azo-CA4 (ESI Fig. S8†), and therefore must be due to
a rearrangement product or products. (Note also that the
samples were irradiated with light immediately prior to injec-
tion which would have eliminated the majority of the trans
isomer even if was present beforehand). Additionally, transient
formation of a peak of 624 Da was observed during the reac-
The half-life of thermal reversion of Azo-CA4 and its future
analogs is a critical piece of their potential use as automatic
turn-off drugs and tools.15 It is therefore significant that our
values for the half-lives of Azo-CA4 vary significantly from
those determined by Borowiak et al. under the same con-
ditions; our value is about 14-fold longer (85 min vs.
6.2 min).30 Although in cells the half-life of Azo-CA4 can be
altered by thiols, the cellular environment,15 and by tubulin
binding itself, all of our cell culture data supports the longer
half-life value. For example, our measured EC50 values for Azo-
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Fig. 3 Analysis of the inhibition of Azo-CA4 in cell culture. Cells were treated with varying concentrations of Azo-CA4 for 48 hours and were either
exposed to 380 nm light (4.4 mW cm−2) (closed circles) or kept in the dark (open triangles). In (a) HUVEC cells were treated with light for 1 min every
2 h. In (b) MDA-MD-231 cells were treated with light for 1 min every 1 h. In (c) HUVEC cells were treated with 1 minute light pulses at varying inter-
vals: no light (black circles), 1 × 1 minute at the beginning of the experiment (green triangles), 24 × 1 minute (every 2 h), and 48 × 1 minute (every
1 h). Cell viability assays reported as a percentage of vehicle controls. The value in parentheses is the EC50. In (d), the cells were treated with 380 nm
light (4.4 mW cm−2, 1 minute every hour) or kept in the dark for 48 h followed by analysis of cell density with Cell Titer Blue.
Table 1 EC50 values of cell viability assays with Azo-CA4 and CA4
MDA-MB-231a
HUVECb
HUVECc
HUVECd
Azo-CA4 light
Azo-CA4 dark
CA4
0.6 0.07 μm
21.1 0.6 μm
0.0037 0.2 μm
0.4 0.03 μm
0.7 0.04 μm
—
—
7.7 1.5 μm
—
—
9.5 0.8 μm
0.0021 0.2 μm
a Cell viability assay with light pulse for 1 min every 1 hour. Error represents triplicate experiments. b Cell viability assay with light pulse for 1 min
every 1 hour. Error represents 5 wells. c Cell viability assay with light pulse for 1 min every 2 hours. Error represents triplicate experiments. d Cell
viability assay with light pulse for 1 min at beginning of experiment Error represents 5 wells.
CA4 with MDA-231 cells are very similar to those determined
Glutathione reactions with azobenzenes have been studied
by Borowiak et al. on this cell line,30 yet we pulsed with light on a number of systems. For most azobenzenes, nucleophilic
once per hour, and they pulsed every 15 seconds. If the half- attack of GS− anion on the diazo nitrogen gives the intermedi-
life was 6.2 minutes as reported,30 we should have observed ate sulfenylhydrazide45 (Scheme 2). The sulfenyl hydrazide is
significantly weaker potency with our pulse regimen.
then either attacked by a second glutathione anion to displace
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Fig. 4 Glutathione exposure in light. Azo-CA4 (300 μM in 5.7% DMSO/PBS pH 7.2) in the presence of fully reduced glutathione (10 mM in PBS pH
7.2) and TCEP (5 mM in PBS pH 7.2) was placed in 3 separate wells (100 μL per well) of a 96 well plate at 37 °C and (a) exposed to light (380 nm,
4.4 mW cm−2) for 1 min every 2 h. Absorbance spectra were taken at different time points directly preceding exposure to light and are expressed as
an average of triplicate readings. (b) Is identical to (a) except the reaction was kept in the dark, and the Azo-CA4 concentration was 400 μM. (c) and
(d) plot the change absorbance of 380 nm over time of the data in (a) and (b) compared to identical reactions lacking glutathione. The decrease in
absorbance of the minus GSH control in (c) is due to incomplete relaxation back to the trans state.
the reduced hydrazide (Scheme 2) or is hydrolyzed by water to logical pH. Moreover, we did not see any ions corresponding
give back the azobenzene plus a sulfenic acid. The hydrazide is either to the mass of the phenylhydrazide or the sulfenylhydra-
easily oxidized back to the azobenzene, and therefore, the net zide during the course of the reaction. Instead, our LC-MS
result of each of these reactions is re-formation of the original data shows that a transient adduct of Azo-CA4 and GSH with a
trans-azobenzene. By a similar mechanism, glutathione cata- mass of 624 is formed during the GSH reaction. This mass is
lyzes the relaxation of cis-azobenzenes back into their trans two less than would be expected for the sulfenyl hydrazide.
form.15,46 With Azo-CA4; however, our data shows that reaction The formation of this 624 Da species could potentially be
with glutathione leads to the destruction of the azobenzene explained by nucleophilic attack of the azo nitrogen onto GSSG
structure. This has been observed before by Woolley with other to form the sulfenyl diazo cation. To test this, we incubated
electron rich methoxy-substituted azobenzenes.41,42,47 Woolley Azo-CA4 with GSSG, but no reaction was observed over 18 h
proposed that the enhanced basicity of the electron rich azo (data not shown) which rules out this potential mechanism.
nitrogens leads to protonation which primes the compound One could envision other possibilities for formation of the sul-
for nucleophilic attack by GSH. However, this proposed mech- fenyl diazo cation, or loss of the two hydrogens via a mechan-
anism leads to the production of the same sulfenyl hydrazide ism involving electrocyclization of the two rings. Since the
(Scheme 2) that would be expected to result in reversion back light-activated Azo-CA4 degrades more rapidly than that in the
to the azobenzene. We measured the pKa of Azo-CA4, and it is dark (Fig. 4c), photochemical pathways for degradation that
below 4.6 (ESI Fig. S10†), so it will not be protonated at physio- also involve GSH could also be envisioned. More work needs to
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Fig. 5 ESI-MS analysis of the reaction of glutathione and Azo-CA4. Azo-CA4 (300 μM in 5.7% DMSO/PBS pH 7.2) in the presence of fully reduced
glutathione (10 mM in PBS pH 7.2) and TCEP (5 mM in PBS pH 7.2) was placed in 3 separate wells (100 μL per well) of a 96 well plate at 37 °C and
exposed to light (380 nm, 4.4 mW cm−2) for 1 min every 2 h immediately preceding analysis. At the times given, a sample was removed and injected
onto an analytical column containing 5 μm phenyl C-18 YMC and analyzed by ESI-MS. (a) Traces for ions of M + H identical to Azo-CA4 (319 Da). (b)
Traces corresponding to m/z of 624 Da. Slight retention time drifts are expected due to manual loading of the capillary containing sample onto the
instrument (see Fig. S7† for the LC-MS spectra of co-injected standards).
Acknowledgements
The authors are grateful to the Ohio Supercomputer Center for
a generous allocation of computational resources. This work
was supported by grants awarded to M.C.T.H. (R15CA167582)
and J.C.H. (R01GM092827) from the National Institutes of
Health. We thank Dr. Richard Moran, Dr. Massimo Bertino,
David Hacker, Erica Peterson, Samantha Katner, and Dr. Stacie
Richardson for experimental assistance and scientific
discussion.
Scheme 2 Reactions of azobenzenes with glutathione. Reported reac-
tion cycle of azobenzenes with glutathione.
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