Photoactivated cell-killing involving a low molecular weight, donor-acceptor diphenylacetylene

Article abstract of DOI:10.1039/c9sc00199a

Photoactivation of photosensitisers can be utilised to elicit the production of ROS, for potential therapeutic applications, including the destruction of diseased tissues and tumours. A novel class of photosensitiser, exemplified by DC324, has been designed possessing a modular, low molecular weight and 'drug-like' structure which is bioavailable and can be photoactivated by UV-A/405 nm or corresponding two-photon absorption of near-IR (800 nm) light, resulting in powerful cytotoxic activity, ostensibly through the production of ROS in a cellular environment. A variety of in vitro cellular assays confirmed ROS formation and in vivo cytotoxic activity was exemplified via irradiation and subsequent targeted destruction of specific areas of a zebrafish embryo.

Full text of DOI:10.1039/c9sc00199a

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DOI: 10.1039/C9SC00199A
Photoactivated cell-killing involving a low molecular
weight, donor-acceptor diphenylacetylene†
David R. Chisholm,a Rebecca Lamb,b Tommy Pallett,bc Valerie Affleck,d Claire Holden,ab
Joanne Marrison,e Peter O’Toole,e Peter D. Ashton,e Katherine Newling,e Andreas
Steffen,f Amanda K. Nelson,f Christoph Mahler,f Roy Valentine,g Thomas S. Blacker,h
Angus J. Bain,h John Girkin,∗c Todd B. Marder,∗ f Andrew Whiting∗a and Carrie A.
Ambler∗b
Photoactivation of photosensitisers can be utilised to elicit the production of ROS, for potential
therapeutic applications, including the destruction of diseased tissues and tumours. A novel class
of photosensitiser, exemplified by DC324, has been designed possessing a modular, low molec-
ular weight ‘drug-like’ structure which is bioavailable and can be photoactivated by UV-A/405 nm
or corresponding two-photon absorption of near-IR (800 nm) light, resulting in powerful cytotoxic
activity, ostensibly through the production of ROS in a cellular environment. A variety of in vitro
cellular assays confirmed ROS formation and in vivo cytotoxic activity was exemplified via irradi-
ation and subsequent targeted destruction of specific areas of a zebrafish embryo.
1 Introduction
type I and type II. A type I process can occur either from the S1
or the T1 state of the photosensitiser and involves either hydro-
gen or electron transfer between the excited photosensitiser and
a substrate. The resulting radicals can react with molecular oxy-
gen (3O2), typically producing ROS species such as superoxide
(O·2– ) and hydroxyl radicals (OH·). A type II process involves di-
rect energy transfer from the longer-lived T1 state of the excited
The generation and modulation of reactive oxygen species (ROS)
is of huge importance in the control, maintenance, defence and
death of both eukaryotic and prokaryotic cells.1,2 Indeed, ROS
are produced by a number of biochemical processes in order to
modulate cellular behaviour; however, they can also be gener-
ated by the action of the excited states of chemical compounds
known as photosensitisers formed through absorption of light.3
Light activation of a photosensitiser typically involves photoexci-
tation from the ground state (S0) to a singlet excited state (S1),
which can then undergo intersystem-crossing to a triplet excited
state (T1).4,5 ROS formation can then occur via two pathways,
3
photosensitiser to O2, generating singlet oxygen (1O2).6
In a cellular context, these oxygen-derived species elicit a va-
riety of modulatory effects depending on the rate and extent of
their production; at high concentrations apoptosis is observed,
while at low concentrations a stimulatory response is often evi-
dent.7–9 Both pathways hold considerable therapeutic potential,
but the majority of photosensitisers in the clinic are used for pho-
todynamic therapy (PDT), in which a photosensitiser is excited
near/inside a particular target tissue or condition (e.g. micro-
bial infections, neoplasias, tumors, etc.), causing the generation
of large quantities of ROS and subsequent destruction of that
tissue.3,10 Photosensitisers clinically approved for PDT of vari-
ous cancers include Photofrin® (Figure 1) and 5-aminolaevulinic
acid, which is metabolized to the active photosensitiser, protopor-
phyrin IX.11–13
a
Department of Chemistry, Durham University, Science Laboratories, South Road,
Durham DH1 3LE, UK
b
Department of Biosciences, Durham University, South Road, Durham, DH1 3LE, UK
c Biophysical Sciences Institute, Department of Physics, Durham University, South Road,
Durham, DH1 3LE, UK
d
LightOx Limited, Wynyard Park House, Wynyard Avenue, Wynyard, Billingham, TS22
5TB, UK
e
Bioscience Technology Facility, Department of Biology, University of York, York, YO10
5DD, UK
f
Institut für Anorganische Chemie, Julius-Maximilians-Universität Würzburg, Am
However, these compounds exhibit inadequate pharmacologi-
cal properties including poor dosage control due to the require-
ment for metabolic processing, and extremely long biological half-
lives, which causes skin photosensitivity for weeks after treat-
ment.3 Indeed, the vast majority of recently reported photosen-
sitizers are high molecular weight, hydrophobic, often metal-
Hubland, 97074 Würzburg, Germany
High Force Research Ltd., Bowburn North Industrial Estate, Bowburn, Durham, DH6
g
5PF, UK
h
Department of Physics & Astronomy, University College London, Gower Street, Lon-
don, WC1E 6BT, UK
†
Electronic Supplementary Information (ESI) available.
See DOI:
10.1039/b000000x/
1–11 | 1

Chemical Science
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DOI: 10.1039/C9SC00199A
R
NH
O
N
OR2
O
N
O
H
N
DC324: R1 = iPr, R2 = H
DC473: R1 = propargyl, R2 = Me
O
N
R1
NaO2CH2C
n = 1-6
Photofrin®
DC324 and DC473
KillerRed
! Potent photosensitiser
" Protein: 27 kDa
! Potent photosensitisers
! Small molecules: 0.4 kDa
! Facile, modular synthesis
! Drug-like
! Potent photosensitiser
" Polymer: 1 to 7 kDa
" Long biological half life
" Not an ideal drug
" Genetically encoded
" Not a viable drug
Fig. 1 Comparison of the characteristics of DC324 and DC473 with those of existing photosensitisers KillerRed and Photofrin®
containing porphyrin, chlorin and phthalocyanine scaffolds.14
The large size of these molecules has a significant and negative ef-
fect on cell permeability and diffusion rate into and out of the cell,
and non-specific cell death is another major problem due to both
the inability to target the compounds locally and issues with ac-
cumulation in off-target tissues and organs.3,15,16 Therefore, the
inherent properties of these structures make them efficient pho-
tosensitisers, but far from ideal as therapeutics. A genetically en-
coded variant of green fluorescent protein (GFP), KillerRed (Fig.
1), is also a potent photosensitiser; however, the requirement for
genetic modification probably makes KillerRed unviable for clin-
ical PDT, though the method has great potential for biological
studies.17 Due to these drawbacks, fine control of the release of
ROS in a therapeutic context is not possible with current photo-
sensitisers. Consequently, there is a major need for novel com-
pounds and ROS generation mechanisms that are able to elicit
an entirely controlled either destructive or proliferative cellular
response based on the requirements of the disease, and indeed,
the temporal requirements of clinical treatment. In this paper,
we report a new class of novel, low molecular weight, drug-like
compounds which exhibit the potential to elicit such controlled
cytotoxic activity.
was synthesised by the coupling of 6-iodo-tetrahydroquinoline
(reported previously34) 5, with acceptor alkyne 4 (Figures 2 and
3).
O
O
HBF4
NaNO2
NH2
N2BF4
O
O
CaCO3
Pd(OAc)2
MeOH
H2O
0 oC, 1 h
83%
2
I
I
I
1
RT, 1.5 h
75%
TMS
Pd(PPh3)2Cl2
CuI, Et3N
RT, 16 h
83%
O
O
O
TBAF
THF
O
-20 oC, 1 h
95%
4
3
TMS
Fig. 2 Synthesis of acceptor alkyne 4.
Compound 4 was synthesised via a four-step approach, begin-
ning with diazotisation of 4-iodoaniline to give the bench-stable
diazonium tetrafluoroborate 1. This was suitably reactive un-
der Heck-Matsuda conditions,36 enabling effective conversion to
the desired cinnamate 2 in 75% yield with complete E-selectivity
(crystal structure shown in the ESI†). Sonogashira coupling with
trimethylsilylacetylene gave the protected alkyne 3,37 which was
initially deprotected using K2CO3 in MeOH/DCM to give the tar-
get acceptor alkyne 4. While these conditions did remove the
TMS protecting group, it was also found that the presence of
traces of EtOH present in the commercial grade of MeOH used
for the reaction caused efficient transesterification to the corre-
sponding ethyl ester. To circumvent this, deprotection using TBAF
in THF at -20 oC cleanly provided 4 with the methyl ester intact.
Sonogashira coupling of acceptor 4 with donor 5 proceeded with
complete conversion to the desired coupled product which, upon
saponification, gave the target compound DC324 in a 67% yield
2 Results and discussion
2.1 Synthesis
Inspired by our work on the design, synthesis and applications
of synthetic retinoids for controlling cellular development,18–25
we recently designed fluorescent analogues of such compounds
for use in fluorescence-based biophysical studies.26,27 It is known
that the addition of a strong π-donor (-NR2) and strong π-
acceptor (-CO2R) to a diphenylacetylene scaffold results in ef-
ficient charge transfer from the donor moiety to the accep-
tor moiety upon photoexcitation.28–33 The resulting structures
exhibit strong, solvatochromatic fluorescence with significant
bathochromic shifts in absorption and emission spectra in polar
media due to the efficient formation of intramolecular charge
transfer (ICT) excited states, and we were interested in synthesis-
ing a range of such compounds that incorporated highly lipophilic
and electron rich tetrahydroquinoline donor structures34,35 for
use in cellular imaging studies. One such compound, DC324,
O
1. Pd(PPh3)2Cl2
CuI, Et3N
RT, 72 h
OH
O
I
O
+
N
2. 20% NaOH
THF, reflux
40 h
DC324
4
5
N
67% over three steps
3. 5% HCl
Fig. 3 Coupling of acceptor alkyne 4 and donor tetrahydroquinoline 534
to give DC324.
2 |
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Table 1 Photophysical properties of DC324 in a variety of solvents, MeCy
= methylcyclohexane.
to excite larger numbers of molecules, thus reducing the risk of
thermal damage to the tissue and also enables deeper penetration
in more scattering tissue samples.
Solvent
MeCy
λ
abs(max)/nm (ε/M-1cm-1)
394, 296
λ
em(max)/nm
φ
-
τ/ns
-
The most interesting finding, however, was that when DC324
and DC473 were irradiated with UV-A (350-390 nm) light in cul-
tured epithelial cells, rapid cell death was observed that was,
possibly, consistent with the production of large amounts of ROS
(Fig. 4D/E).
434, 460
498
513
533
Toluene 395(30600), 297(27700)
0.92 2.0
MeCN
EtOH
DMSO
H2O
385(31200), 295(28000)
366(41100), 294(30800)
380(32500), 297(32200)
385, 298
0.04
0.10
0.30
-
-
-
-
-
-
512
562
622
CH2Cl2
403, 301
-
2.3 In vitro characterisation
over the three steps. We have also recently reported the syn-
thesis, fluorescence properties and cellular localisation behaviour
of DC473 (Figure 1), a close analogue of DC324 with a methyl
cinnamate acceptor group and N-propargyl tetrahydroquinoline
donor which can be utilised for dual fluorescence/Raman imag-
ing in cells.38
To investigate this observed cell death we performed a range
of cellular and cuvette-based experiments to understand locali-
sation and mode of action. According to confocal fluorescence
microscopy, DC324 was found to exhibit non-specific localisation
in non-polar, membrane-rich environments, including mitochon-
dria (Pearson’s correlation with MitoTracker Red, R = 0.81) and
other organelles (Fig. 5A).38 This finding was supported by the
fact that the emission peaks (λex = 405 nm) detected at 460-490
nm were similar to cuvette-based measurements in non-polar sol-
vents such as toluene or methylcyclohexane. However, the ob-
served emission wavelength varied subtly (Figure 5B) according
to the cellular environment, as a result of the solvatochromatic
behaviour of the compound.
We next utilised the redox reactive dye CellRox,40 which flu-
oresces in response to oxidation by ROS, to determine whether
ROS is produced either by, or in response to, in cellulo photoac-
tivation of DC324. Whereas in the absence of the compound no
CellRox fluorescence (and no cell death) was observed, DC324-
treated cells subsequently activated with 405 nm light exhibited a
strong CellRox fluorescence signal with a steady increase immedi-
ately following irradiation, particularly in intracellular organelles
(Fig. 6). Similar behaviour was observed in DC473-treated cells
and, therefore, this initially suggested that the compounds may
act as photosensitisers to elicit ROS formation in cells.
2.2 Photophysical characterisation
Photophysical and computational studies showed that DC324 ex-
hibits two strong absorptions (Table 1, Fig. 4A) with extinction
coefficients of ca. 27,000-41,000 M-1 cm-1 in the UV and violet
regions, respectively, both arising from the formation of ICT ex-
cited states. Consequently, a solvatochromatic absorption was ob-
served, wherein polar solvents give rise to a hypsochromic shift,
which indicates that the ground state S0 is more polar than the
Franck-Condon S1 excited state. However, even more impressive
is the highly solvatochromatic fluorescence (Table 1, Fig. 4B),
with quantum yields of up to 0.92 in non-polar solvents such as
toluene,28,29 and an emission lifetime of 2.0 ns. However, in
more polar solvents, such as EtOH, DMSO or CH2Cl2, a second
excited state process occurs, giving rise to the reproducible obser-
vation of a secondary decay starting around 500 ps after the laser
pulse, which was observed on several spectrophotometers and is
not an artifact of a particular instrument. DC324 shows similar
photophysical properties when compared to DC47338 although
DC324 exhibits bathochromic shifts in absorption and emission
in non-polar solvents, presumably due to the increased donor
strength of the N-iPr moiety in comparison to the N-propargyl
group of DC473. We also measured the two-photon absorption
(TPA) spectra of DC324 and DC473 in toluene (Fig. 4C), and
note the TPA cross-sections of around 500 GM at 790 nm which
are impressive for such a small dipolar molecule. Comparing
with, for example, a simple dimethylamino donor/dimesitylboryl
acceptor-substituted diphenylacetylene,39 which has similar ab-
sorption properties in toluene, the emission spectra of DC324 and
DC473 are somewhat red-shifted, and the TPA cross-section is ca.
2.5 times higher for the latter two compounds. This can likely be
attributed to a combination of the increased donor strength of the
rigidified tetrahydroquinoline-based moiety and the extension of
the conjugation length via the alkene unit in DC324 and DC473.
The TPA cross-section is the typical measure of the efficiency with
which a compound absorbs two photons; for one photon absorp-
tion, the efficiency of absorption is typically reported as an ex-
tinction coefficient. In a cellular and biological imaging context,
a greater TPA cross-section allows lower laser powers to be used
Because of the structural relationship with retinoids such as
all-trans-retinoic acid (ATRA) and other synthetic retinoids,20 we
were interested to determine whether retinoids, in general, elicit
ROS production when cells are treated with light. We, therefore,
chose EC23 (Fig. 7A),18 an analogue of DC324 and DC473 with
retinoid biochemical activity but no absorption or photoactivity at
405 nm (since it lacks the amine donor functionality), to act as
a negative control. In these experiments, treatment of cells with
EC23 and subsequent irradiation did not cause activation of Cell-
Rox nor any observable cell death (Fig. 7B). It was, therefore,
clear that the photophysical properties of DC324 and DC473, i.e.
the ability to absorb light of 405 nm due to the addition of strong
π-donor and extended π-acceptor moieties, were mandatory to
initiate cell death upon photoactivation rather than an inherent
biochemical or biological signalling activity of a diphenylacety-
lene retinoid or retinoid-like structure. Furthermore, we have
previously shown that retinoid signalling activity can be com-
pletely eliminated when the compound is of a length greater than
EC23/ATRA and, hence, given that DC324 and DC473 fit this non-
retinoid structural criterion, we can be confident that the pho-
toactivated cell killing activity is not related to retinoid signalling
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A
B
C
D
E
Fig. 4 A) Absorption spectra of DC324 in a variety of solvents and electron density difference plots for the two observed major absorption bands
S0 → S1 (low energy) and S0 → S2 (high energy) obtained from TD-DFT calculations at the M06-D3-BJ/def2-TZVP level of theory. B) Emission spectra
of DC324 in a variety of solvents. C) Two-photon absorption spectra of DC324 and DC473 in toluene. D) HaCaT keratinocytes treated with 10 µM
DC324 for 30 min, prior to irradiation. E) HaCaT keratinocytes treated with 10 µM DC324 after multiple short irradiations with 405 nm, 12 hours after
initial imaging.
processes.21
cated by a secondary process starting at around 500 ps after light
excitation. Alternatively, when bound to a cellular membrane
(e.g. mitochondrial), a conformational change in the photoex-
cited state of the compound may induce signicant disruption of
this membrane and, therefore, the release of ROS as a biological
response to this cellular stress. In either case, our experiments
clearly show that the processes responsible for the observed cell
death upon photoexcitation of the compounds are not trivial.
In order to identify whether DC324 and DC473 directly cause
the formation of a specific ROS upon photoactivation, we per-
formed a variety of cuvette-based photophysical experiments in-
volving solutions of the compounds dissolved in a range of oxygen
saturated solvents, at various concentrations, with irradiation at
both absorption maxima (experimental details in ESI†). These in-
cluded 1O2 sensitisation and phosphorescence detection thereof,
hydroxyl radical detection by absorption measurements of added
methylene blue, peroxyl radical detection by fluorescence mea-
surements of added rhodamine 6G, and hydrogen peroxide de-
tection by chemiluminescence of added luminol in the presence
of a cobalt(II) catalyst (the latter three types of experiment were
carried out with the addition of H2O to allow for facile formation
of ROS from H2O). None of these experiments returned a positive
result. Furthermore, there was no evidence, either through exper-
imental or theoretical means, that the compounds are capable of
generating triplet excited states to any significant extent.
We next employed RNA sequencing to provide information on
the biological and cellular responses that occur following in cel-
lulo photoactivation of the compounds. This higher level ap-
proach enables ‘cause-and-effect’ examination of the genes and
genetic pathways that are regulated in response to a stimulus.
Keratinocytes were treated with 1 µM DC473 for 4 hours and
were subsequently irradiated with UV-A (363-385 nm) light for 1
minute. After 1 hour, RNA was collected from cells that had been
DC473-treated and irradiated, and from corresponding DMSO-
treated and DC473-treated, non-irradiated controls. Gene set
enrichment pathway analysis (see ESI†) of these RNA samples
indicated that, among other effects, genetic responses towards
the presence of oxygen species were significantly upregulated
when cells treated with DC473 were irradiated in comparison
to those of non-irradiated DC473-treated cells, and both irradi-
ated and non-irradiated DMSO-treated cells. Responses towards
the generation of nitrogenous compounds were also evident, per-
haps indicating the generation of nitric oxide - a radical species
that is heavily implicated in oxidative stress pathways resulting
from PDT.41,42 Furthermore, gene regulatory pathways impli-
cated in cell death processes were only upregulated in the ir-
Given the fact that we can, nevertheless, detect ROS upon pho-
toexcitation of DC324 and DC473 in cells using CellRox, it is clear
that an alternative ROS generation mechanism may be taking
place. This could involve changes of the excited state properties of
DC324 and DC473 and thus ROS formation by direct association
with a second species of cellular origin, or that the photosensitis-
ing process involves sensitisation of another species by interaction
with the compounds upon light activation. Such complex excited
state modes of action would be in line with our previous observa-
tion that in an environment where hydrogen bonding is possible,
i.e. in polar solvents, the excited state lifetime decay is compli-
4 |
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A
B
1.0
0.8
0.6
0.4
0.2
0.0
Average ROI
ROI 1
ROI 2
DC324
DMSO
Fig. 6 Detection of fluorescence produced by ROS reactive dye, CellRox
in HaCaT keratinocytes treated with 1 µM DC324 or a DMSO control.
Scale bars equal 25 µm.
400
500
600
700
Emission Wavelength (nm)
DC324
C
dited and increased the extent of cell death (Fig. 8B). The cy-
totoxic effect of the compound was also characterised by a dose-
response relationship between the cell viability and the treatment
concentration. Accordingly, we determined an EC50, based on
the average viable fraction of the epithelial cellular population
24 hours after the initial irradiation event, of 0.20 ± 0.01 µM
(Fig. 8C). The time required to induce cell death, and the change
in physiology, were also significantly affected by compound con-
centration, i.e. irradiated cells exposed to the higher concentra-
tions tested (10 µM) induced necrosis-like cell death in less than
60 seconds whilst cells exposed to lower concentrations (e.g. 1
µM) showed an initial cell membrane blebbing associated with
cell apoptosis. Cell loss was rapid, and maximal impact on cell
viability was detected approximately 400 minutes after the ini-
tial irradiation event. Importantly, cells exposed to the compound
without irradiation, or irradiation treatment alone, had no impact
on cell viability. These physiological and morphological changes
are consistent with cell death in response to the presence of sig-
nificant amounts of ROS;44 however, given the apparent inability
for DC324 and DC473 to access triplet states that could lead to
the generation of ROS through the paradigm set out by the por-
phyrins and other similar compounds,5 it is clear that an alterna-
tive cytotoxic mechanism may be operating. Whether this effect
is achieved through sensitisation of a second species by the pho-
toactivated compound or, for example, through disruption of the
membrane/organelle(s) which the compound is bound to or via
another biological response is unclear, and the enormous com-
plexity of a cellular system makes interrogation of this effect in-
herently difficult. Nonetheless, as an orthogonal means for the
generation of a photodynamic therapeutic effect, the unique ac-
tivity of DC324 and DC473 is highly compelling and of great po-
tential utility.
#"!$%
#"!$
MitoTracker
Fig. 5 (A) HaCaT keratinocytes treated with 1 µM DC324 (green) and
co-stained with MitoTracker Red (red). Scale bars equal 50 µm. (B) Peak
emission wavelength of DC324 in regions of interest (ROI) of cells, with
excitation at 405 nm. (C) ROI 1, green circle marks area distal to nuclear
membrane (λmax = 490 nm) and ROI 2, blue circle, marks area adjacent
to nucleus (λmax = 460 nm). Average emission wavelength for red square
was calculated (Average ROI λmax = 475 nm).
radiated, DC473-treated cells. While these experiments do not
provide a precise molecular understanding of the reactive species
that are generated, they do provide strong and, in many respects,
more direct evidence that oxygen-containing species (and other
nitrogenous species) are produced upon in cellulo photoactiva-
tion of DC324 and DC473, and that this phenomenon is likely to
be the cause or at least related directly to the observed cell death
activity.
In vitro cultured epithelial, mesenchymal and colorectal (see
ESI†) cancer cells were next treated with a range of concentra-
tions of DC324, and a variety of experiments were conducted
in order to further quantify and characterise the observed light-
activated cytotoxic activity. In all these human cell lines, irradia-
tion with either broadband UV-A (363-385 nm), or a monochro-
matic 405 nm light source induced rapid cell death. Furthermore,
cell death was also observed when the compound was irradiated
at the corresponding two-photon absorption wavelengths (800
nm), a particularly important property, as this enables deeper tis-
sue penetration (see ESI† for irradiation images).43 It was fur-
ther found that the cell death timeline could be modulated by
varying the irradiation area, photon flux and compound concen-
tration. Figure 8A illustrates that only the area which received
UV-A irradiation was destroyed, highlighting that the cell-killing
activity of the compounds is a light-activated effect, rather than
one of general cytotoxicity. Furthermore, the amount of energy
supplied was important; stronger irradiation per unit area expe-
2.4 In vivo characterisation
To test the light-activated cytotoxic activity in an in vivo context,
48-hour old wild type zebrafish embryos were exposed to 1 µM
DC324 for 2 hours, then transferred to E3 water without the com-
pound and incubated for 4 hours before irradiation. Embryos ir-
radiated (UV-A; 28 J/cm2) in the developing tail region showed
evidence of cell death in the irradiated area but not in adjacent
regions.45 Following irradiation, nuclei became distended and ir-
1–11 | 5

Chemical Science
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O
OH
A
B
0 s
EC23
25
20
15
10
5
1.0
0.8
0.6
0.4
0.2
0.0
'&$#%
(&#$
+01.92;0
320 s
640 s
0
-200
0
200
-240"8
400
600
Fig. 7 (A) Chemical structure of synthetic retinoid, EC23. (B) Quantitation of relative fluorescence of CellRox before and after irradiation using a 405
nm laser. The relative fluorescence intensity was calculated in the region of interest (ROI, yellow box) at each time point and data graphed using
box-whisker plot. Graph coloured lines denote sample mean (n = 11, DC324; n = 3, EC23; n = 3, negative) from 2 experimental replicates for each
treatment (red = DC324, pink = EC23, blue = negative). Time 0 equals time of irradiation. Increased fluorescence intensity in DC324 treated cells was
significant as determined by one-way ANOVA (p<0.001). Scale bars equal 50 µm.
regular in shape, a common characteristic of nuclear apoptosis
(Fig. 9).46 Together, these data clearly show that DC324 and
DC473 have the potential to act as a potent modality for the de-
struction of tissue in vitro and in vivo through a light-activated
cytotoxic effect.
eased tissues, or to another small molecule to influence subcel-
lular localisation. The small molecular footprint of these novel
photosensitisers would likely have a negligible effect on the effi-
cacy of these targeting modalities, which is often not the case,
for example, with antibody-conjugated porphyrin-based photo-
sensitisers.49,50 Hence, the advantages of this new class of small,
adaptable organic PDT agent, which is highly cell-permeable, po-
tent and appears to exhibit a unique mode-of-action, could prove
to be the key to realising the rich potential PDT has for tackling
a wide range of disease treatment applications which, to date,
has been severely limited by the inadequate properties of existing
photosensitisers.
3 Conclusions
A class of small, organic, conjugated compounds, essentially ho-
mologues of synthetic retinoids, show clear potential as a novel
photosensitising agent. DC324 and DC473 possess a much more
‘drug-like’ molecular weight and structure in comparison to ex-
isting photosensitisers and a thorough biological characterisation
has exemplified the ability for these compounds to elicit cyto-
toxic activity, ostensibly through bringing about robust intracel-
lular ROS production following UV-A or violet light absorption,
or corresponding two-photon absorption via near-IR irradiation.
While UV-based photodynamic therapies have typically seen only
limited use in the past,47,48 in more recent years significant ad-
vances in optical and endoscope technology have enabled the
production of devices including low cost portable LED-based il-
lumination systems capable of precisely targeted irradiation at a
specific wavelength. These next generation optical devices are be-
ginning to see widespread use in the clinic and, hence, potentially
pave the way for a treatment modality involving UV-activated,
drug-like photosensitisers in previously impractical or impossible
treatment regimes. However, the two-photon absorption ability
of the compounds also paves the way for use in those contexts
where UV-activation remains intractable, taking advantage of the
greater tissue penetration of near-IR irradiation. Furthermore,
the modular structures of DC324 and DC473 could be modified
in further studies to incorporate targeting mechanisms, such as
conjugation to an antibody to enable specific localisation to dis-
4 Experimental
4.1 General Synthetic Information
Reagents were purchased from Sigma-Aldrich, Acros Organics,
Alfa-Aesar and Fluorochem. Reagents were purified, if required,
by recrystallisation or distillation/sublimation under vacuum.
Solvents were used as supplied from Fisher Scientific or Sigma
Aldrich, and dried before use if required with appropriate drying
agents or using an Innovative Technologies Inc. Solvent Purifi-
cation System. Thin-layer chromatography (TLC) was conducted
using Merck Millipore silica gel 60G F254 25 glassplates and/or
TLC-PET foils of aluminium oxide with fluorescent indicator 254
nm (40 × 80 mm) with visualisation by UV lamp or appropriate
staining agents. Flash column chromatography was performed
using SiO2 from Sigma-Aldrich (230-400 mesh, 40-63µM, 60 Å),
or activated neutral aluminium oxide (Alumina) from Sigma-
Aldrich, and monitored using TLC. Sublimation/distillation was
performed using a Buchi Glass Oven B-585 Kugelrohr operating at
a pressure between 0.2-2.0 Torr. NMR spectra were recorded us-
ing Varian VNMRS-700, Varian VNMRS-600, Bruker Avance-400
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100
C
A
B
Irradiated
zone
100
75
50
25
0
75
50
25
0
Control
14 J/cm2
28 J/cm2
42 J/cm2
Non-irradiated
zone
0
200 400 600 800 1000
-4
-3
-2
-1
0
1
2
Time after initial exposure (min)
Concentration uM (log)
Fig. 8 (A) Phase contrast image of confluent sheet of HaCaT epithelial cells exposed to 10 µM DC324 and the righthand area of the field irradiated
with UV-A. Image captured 10 minutes post irradiation; note that cell death occurred only in the irradiated zone. Scale bars equal 100 µm. (B) Cell
viability after irradiation with varying UV-A doses. Dose-response curves have been fitted to the data in a box-whisker plot graph using 4 experimental
replicates (n=4). Statistical significance was determined by one-way ANOVA (p < 0.0001) and Dunnett’s multiple comparison test comparing individual
dose response curves to unirradiated controls (0 J/cm2). The energy fluence of 14, 28 and 42 J/cm2 were considered statistically significant with p
values of 0.0034, 0.0002 and < 0.0001, respectively. (C) The average viable fraction of HaCaT epithelial cellular populations 24 hours after an initial 60
seconds UV-exposure, as a function of the concentration of compound administered. A non-linear regression curve is fitted to the data (experimental
replicates, n = 3; R2 = 0.99).
440 analyser.
B
A
Irradiated zone
4.2 Synthesis
4-Iodobenzenediazonium tetrafluoroborate, 1
4-Iodoaniline (10.95 g, 50 mmol) was added to tetrafluoroboric
acid (48% in H2O, 25 mL), and the suspension was cooled to 0 ◦C
before a solution of NaNO2 (3.79 g, 55 mmol) in H2O (13.7 mL)
was added dropwise with vigorous stirring so as to maintain the
internal temperature below 5 ◦C. After addition, the suspension
was further stirred for 1 h at 0 ◦C before the precipitated solid was
isolated by filtration, washed with cold MeOH and dried to give
a crude brown solid. This was dissolved in a minimal amount of
acetone (ca. 55 mL), to which Et2O was slowly added to pre-
cipitate a yellow solid. This was filtered, washed with cold Et2O
and dried to give compound 1 as a pale yellow solid (13.1 g,
83%): m.p. = 106-108 ◦C (decomposition); 1H NMR (700 MHz,
(CD3)2SO) δ 8.35 (d, J = 9.0 Hz, 2H), 8.43 (d, J = 9.0 Hz,
2H); 13C{1H} NMR (151 MHz, (CD3)2SO) δ 113.6, 115.1, 132.8,
140.2; IR (ATR) vmax/cm–1 3090w, 2282s, 1548m, 1461w, 824s,
523m; MS (EI): m/z = 204 [M−N2]+; Found: C, 22.83; H, 1.30;
N 8.83, Calc. for C6H4BF4IN2: C, 22.67; H, 1.27; N, 8.81%.51
UV-A 0 min
Phase 0 min
C
Phase 40 min
Fig. 9 48 hour wild-type zebrafish embryo treated with 1 µM DC324
for 2 hours prior and the tail irradiated with 42 J/cm2 of UV-A (time = 0
minutes); phase contrast images captured at the time of irradiation (A/B)
and 40 minutes after irradiation (C).
or Varian Mercury-400 spectrometers operating at ambient probe
temperature. NMR peaks are reported as singlet (s), doublet (d),
triplet (t), quartet (q), broad (br), septet (sept), combinations
thereof, or as a multiplet (m), with reference to the following
deuterated solvent signals: CDCl3 (1H = 7.26 ppm, 13C = 77.23
ppm), (CD3)2SO (1H = 2.50 ppm, 13C = 39.50 ppm). ESMS
was performed using a TQD (Waters Ltd., UK) mass spectrometer
with an Acquity UPLC (Waters Ltd., UK), and accurate mass mea-
surements were obtained using a QtoF Premier mass spectrome-
ter with an Acquity UPLC (Waters Ltd., UK). ASAP measurements
were performed using an LCT Premier XE mass spectrometer and
an Acquity UPLC (Waters Ltd., UK). GCMS was performed with a
QP2010-Ultra (Shimadzu) GCMS. IR spectra were recorded using
a Perkin Elmer FTIR spectrometer. Melting points were obtained
using a Gallenkamp melting point apparatus and are uncorrected.
Elemental analysis was conducted using an Exeter Analytical CE-
Methyl (2E)-3-(4-iodophenyl)prop-2-enoate, 2
Pd(OAc)2 (0.138 g, 0.61 mmol), CaCO3 (2.40 g, 24.0 mmol) and
compound 1 (5.54 g, 17.4 mmol) were suspended in MeOH (60
mL). Methyl acrylate (2.16 mL, 24.0 mmol) was added, and the
suspension was stirred vigorously for 1.5 h at RT. The solution
was diluted with DCM, filtered through Celite® and evaporated
to give a crude light brown solid (6.3 g). This was purified by
SiO2 chromatography (hexane:DCM, 1:1, as eluent) to give com-
pound 2 as a white solid (3.74 g, 75%): m.p. = 119-120 ◦C; 1H
NMR (600 MHz, CDCl3) δ 3.81 (s, 3H), 6.44 (d, J = 16.0 Hz, 1H),
7.24 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 16.0 Hz, 1H), 7.73 (d, J
= 8.4 Hz, 2H); 13C{1H} NMR (151 MHz, CDCl3) δ 52.0, 96.7,
118.8, 129.7, 134.1, 138.3, 143.8, 167.3; IR (ATR) vmax/cm–1
3080w, 3000w, 2850w, 1708s, 1636m, 1580m, 1483m, 815s,
493m; MS(ES): m/z = 289 [M + H]+; HRMS (ES) calcd. for
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C10H10IO2 [M + H]+: 288.9726, found 288.9733; Found: C,
sion was diluted with hexane and passed through a Celite®/SiO2
plug (eluting with hexane, then hexane:EtOAc (8:2)). The ex-
tracts were washed with sat. NH4Cl (3×) and brine, dried
(MgSO4) and evaporated to give the intermediate ester as an or-
ange solid (0.7 g). This was dissolved in THF (20 mL), and 20%
NaOH (2 mL) added, and the resultant solution was stirred at re-
flux for 40 h. The mixture was cooled, acidified to pH 1 with 5%
HCl, diluted with EtOAc, washed with sat. NH4Cl, H2O and brine,
dried (MgSO4) and evaporated to give a crude yellow solid which
was recrystallised from MeOH to give DC324 as an orange crys-
talline solid (0.46 g, 67% over three steps): m.p. = 214-215 ◦C
(decomposition); 1H NMR (700 MHz; (CD3)2SO) δ 1.16 (d, J =
6.7 Hz, 6H), 1.22 (s, 6H, H11/12), 1.63 (t, J = 6.0 Hz, 2H), 3.18
(t, J = 6.0 Hz, 2H), 4.14 (sept, J = 6.7 Hz, 1H), 6.54 (d, J = 16.0
Hz, 1H), 6.69 (d, J = 8.7 Hz, 1H), 7.17 (dd, J = 8.7, 2.2 Hz, 1H),
7.29 (d, J = 2.2 Hz, 1H), 7.46-7.51 (m, 2H), 7.58 (d, J = 16.0
Hz, 1H), 7.66-7.72 (m, 2H), 12.41 (s, 1H); 13C{1H} NMR (176
MHz; (CD3)2SO) δ 18.6, 29.7, 31.6, 35.9, 36.1, 46.6, 86.9, 94.0,
106.8, 110.5, 119.5, 125.2, 128.4, 128.8, 130.6, 131.1, 131.2,
133.3, 143.0, 144.5, 167.5; vmax/cm–1 2968w, 2929w, 2870w,
2195m, 1683s, 1622m, 1594s, 1514s, 1421m, 1187m, 837m;
MS(ES): m/z = 374 [M+H]+; HRMS (ES) calcd. for C25H28NO2
[M+H]+: 374.2120, found 374.2118; Found: C, 79.90; H, 7.27;
N 3.65. Calc. for C25H27NO2: C, 80.40; H, 7.29; N 3.75%.
41.86; H, 3.14. Calc. for C10H9IO2: C, 41.96; H, 3.15%.52
Methyl
(2E)-3-4-[2-(trimethylsilyl)ethynyl]phenylprop-2-
enoate, 3
An oven-dried Schlenk flask was evacuated under reduced pres-
sure and refilled with Ar, before Pd(PPh3)2Cl2 (0.217 g, 0.31
mmol), CuI (0.060 g, 0.31 mmol) and compound 2 (3.57 g, 12.38
mmol) were added and the flask sealed with a septum. Et3N
(80 mL) and trimethylsilylacetylene (1.76 mL, 12.44 mmol) were
added and the flask evacuated/filled with Ar again (3×). The
mixture was stirred at RT for 16 h. The solution was diluted with
Et2O, passed through Celite®/SiO2 under vacuum, and evapo-
rated to give a crude brown solid (4.5 g). This was purified
by SiO2 chromatography (hexane:EtOAc, 9:1, as eluent) to give
1
compound 3 as a white solid (2.65 g, 83%): m.p. = 76-78 ◦C; H
NMR (600 MHz, CDCl3) δ 0.26 (s, 9H, 3.81 (s, 3H), 6.43 (d, J
= 16.0 Hz, 1H), 7.43-7.48 (m, 4H), 7.65 (d, J = 16.0 Hz, 1H);
13C{1H} NMR (151 MHz; CDCl3) δ 0.1, 52.0, 96.9, 104.7, 118.8,
125.2, 128.1, 132.6, 134.5, 144.1, 167.4; IR (ATR) vmax/cm–1
2952w, 2898w, 2156w, 1715s, 1634m, 1599w, 1442m, 1171s,
840s; MS(ES): m/z = 259 [M + H]+; HRMS (ES) calcd. for
C15H19SiO2 [M+H]+: 259.1154, found 259.1147.
Methyl (2E)-3-(4-ethynylphenyl)prop-2-enoate, 4
Compound 3 (2.21 g, 8.55 mmol) was dissolved in THF (25 mL),
and cooled to -20 ◦C. Tetrabutylammonium fluoride (1.0 M in
THF) (8.98 mL, 8.98 mmol) was then added dropwise and the
resultant solution stirred at -20 ◦C for 1 h, after which H2O was
added, and the solution extracted with EtOAc (3×). The organ-
ics were washed with brine, dried (MgSO4) and evaporated to
give a crude brown solid. This was purified by SiO2 chromatog-
raphy (hexane:EtOAc, 9:1, as eluent) to give compound 4 as a
4.3 General photophysical information
UV-visible absorption spectra were obtained on an Agilent 1100
Series Diode Array spectrophotometer using standard 1 cm path
length quartz cells.
Excitation and emission spectra were
recorded on an Edinburgh Instruments FLSP920 spectrophotome-
ter, equipped with a 450 W Xenon arc lamp, double monochroma-
tors for the excitation and emission pathways, a red-sensitive pho-
tomultiplier (PMT-R928) and a near-IR PMT as detectors, or on
a Horiba Jobin-Yvon Fluoromax 3 spectrophotometer with single
monochromators for the excitation and emission pathways. The
excitation and emission spectra were corrected using the standard
corrections supplied by the manufacturers for the spectral power
of the excitation source and the sensitivity of the detector. The
quantum yields were measured by use of integrating spheres with
either an Edinburgh Instruments FLSP920 spectrophotometer or
the Horiba Jobin-Yvon Fluoromax 3 spectrophotometer. The lu-
minescence lifetimes were measured using a TCSPC module on
an FLSP980 spectrometer equipped with a high speed photomul-
tiplier tube positioned after a single emission monochromator and
operating with pulsed laser diodes (376 or 274 nm, repetition rate
1-5 MHz, pulse width ca. 200 ps, instrument response function
ca. 500 ps). Decays were recorded to 10000 counts in the peak
channel with a record length of at least 1000 channels. The band-
pass of the monochromator was adjusted to give a signal count
rate of <20 kHz. Iterative reconvolution of the IRF with one de-
cay function and nonlinear least-squares analysis were used to
analyse the data. The quality of all decay fits was judged to be
satisfactory, based on the calculated values of the reduced X2 and
Durbin-Watson parameters and visual inspection of the weighted
and autocorrelated residuals.
1
white solid (1.52 g, 95%): m.p. = 93-95 ◦C; H NMR (600 MHz;
CDCl3) δ 3.18 (s, 1H), 3.81 (s, 3H), 6.44 (d, J = 16.0 Hz, 1H),
7.46-7.51 (m, 4H), 7.66 (d, J = 16.0 Hz, 1H); 13C{1H} NMR
(151 MHz; CDCl3) δ 52.0, 79.4, 83.3, 119.1, 124.2, 128.1, 132.8,
134.9, 143.9, 167.4; IR (ATR) vmax/cm–1 3260m, 2996w, 2946w,
2108w, 1700s, 1634m, 1554m, 1431m, 1206s, 831s; MS (EI):
m/z = 186 [M]+; Found: C, 77.40; H, 5.37. Calc. for C12H10O2:
C, 77.40; H, 5.41%.53 Note: Compound 4 can also be purified by
Kugelrohr sublimation under vacuum (115-125 ◦C, 0.8 Torr).
6-Iodo-4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-
tetrahydroquinoline, 5
Full synthetic details for the synthesis of donor tetrahydroquino-
line 5 are available in a previous report.34
(2E)-3-(4-{2-[4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-
tetrahydroquinolin-6-yl]-ethynyl}phenyl)prop-2-enoic acid,
DC324
Compound 5 (0.61 g, 1.85 mmol) was dissolved in Et3N (12
mL), and the resultant solution was degassed by sonication un-
der vacuum, before the atmosphere was replaced with Ar (5×).
Pd(PPh3)2Cl2 (0.13 g, 0.185 mmol), CuI (0.035 g, 0.185 mmol)
and compound 4 (0.36 g, 1.94 mmol) were then added under Ar.
The resultant suspension was stirred at RT for 72 h. The suspen-
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4.4 Computational Details
trum with an Ocean Optics USB 2000+ Spectrometer (see ESI†).
The irradiation beam was optically restricted to a limited region
within the field of view for most experiments (see ESI†). A well-
defined boundary was achieved using a simple mask in the illu-
mination optical path for the widefield measurements and using
ROI scanning for the laser measurements allowing for interroga-
tion of irradiated and non-irradiated areas in the same field of
view. Cells were imaged for up to 24 h on the microscope with
environmental controls.
Calculations (gas-phase) were performed with the ORCA 3.0.2
program suite.54 Geometry optimisations were carried out with
the B3LYP55–57 functional as implemented in ORCA, and a fre-
quency analysis ensuring that the optimized structures corre-
spond to energy minima. The def2-TZVP58,59 basis set was used
for all atoms together with the auxiliary basis set def2-TZVP/J in
order to accelerate the computations within the framework of the
RI approximation. Van der Waals interactions have been consid-
ered by an empirical dispersion correction (Grimme-D3BJ).60,61
TD-DFT calculations for the first 100 singlet and triplet excited
states were performed with the M06 functional62 and the above
mentioned basis sets. Representations of electron density changes
were produced with orca plot as provided by ORCA 3.0.2 and with
gOpenMol 3.00.63,64
4.7 MitoTracker® staining
DC324-treated cells plated on coverslips were incubated for 30
min with MitoTracker® Red (Thermo Fisher, cat. no. M22425)
prepared according to manufacturer’s directions and diluted in
serum containing media (1 mL, 0.1 µM) then rinsed twice
with PBS (pH 7.2) before incubation and subsequent fixation in
paraformaldehyde/PBS (PFA, 4%), for 5 min. Cells were rinsed in
PBS before mounting coverslips in a polyvinyl alcohol mounting
media.
4.5 Cells
HaCaT human epidermal keratinocytes were purchased from a
commercial supplier (Thermo Fisher) and primary human der-
mal fibroblasts (HDF) were isolated from human patients under
ethical guidelines from National Research Ethics Service.65 Both
cell lines were cultured at 37 ◦C/5% CO2 in DMEM (Gibco cat.
no. 10566, high glucose, GlutaMAX Supplement) with 10% fetal
calf serum and 1× penicillin/streptomycin. For experimental pro-
cedures, cells were seeded (1.2×104 - 5×104 per well) in 24-well
or 6-well tissue culture plates (Corning) or on single or 8-well
chambered coverslips for a minimum of 48 h prior to the start
of an experiment. In some experiments, HaCaT (2 × 104) and
HDF (4 × 104) cells were co-seeded in 8-well chambered cover-
slips. Cells were treated with DC324 (stock concentration 10 mM
dissolved in DMSO) at a range of final concentrations (0.001 µM -
10 µM) in media with 0.1% DMSO. As controls, cells were treated
with 0.1% DMSO or 1 µM EC23, a compound with similar chem-
ical structure to DC324, but no (or negligible) absorption when
excited by UV-A or violet light wavelengths.18 Cells were treated
with the compound for 1-4 h before irradiation.
4.8 ROS quantitation
Cells in separate wells were treated with DC324 or EC23 at 1 µM
concentration (0.1% DMSO) in media and incubated in a tissue
culture incubator. A negative control of 0.1% DMSO in media was
also used. After 2 h, CellRox (Sigma C10422 Deep Red 640/665
nm) at a final concentration of 5 µM was added to the cells and
further incubated for 30 min. Cells were then transferred to the
37 ◦C, 5% CO2 environmental chamber on the Zeiss 880 LCSM.
To quantify ROS production detected by 633 nm excitation of
CellRox dye, background fluorescence emission intensities (655-
675 nm) were captured first in each experimental field for 5 min,
then the field was irradiated with 405 nm and fluorescence levels
(655-675 nm) were captured for an additional 10 min following
irradiation. Zeiss Zen Black and FIJI imaging processing software
were used to calculate the ROI relative fluorescence intensity lev-
els. A minimum of 3 experimental replicates were performed for
each treatment.
4.6 Imaging
4.9 RNAseq and Bioinformatics
Live and fixed cells were imaged using a Zeiss 880 Laser Scanning
Confocal Microscope (LSCM) with Airyscan detection and an en-
vironmental chamber at 37 ◦C and 5% CO2. Fixed cells were im-
aged at room temperature and in air. Samples were excited with
a 405 nm, 594 nm or 633 nm laser and imaged with a Plan-
Apochromat 63×/1.4 Oil DIC M27 objective lens. For irradiation-
induced cell death experiments, cells were irradiated and imaged
on either the Zeiss 880 LSCM or a Zeiss Axio Vert A1, wide field
fluorescence microscope with environmental control chamber us-
ing image corrected 10× or 20× objective lenses designed for live
cell imaging. UV-A filtered irradiation (Axio Vert A1) was car-
ried out using an OSRAM 1× HBO 103 W/2 100 Watt mercury
bulb using DAPI excitation filters with a bandwidth of 335-385
nm and emission bandwidth of 420-470 nm according to manu-
facturer’s specification; 405 nm irradiation (880 LSCM) was car-
ried out with a 405 nm diode laser. Excitation wavelengths for
both microscopes were confirmed by measuring the light spec-
HaCaT human keratinocyte cells were seeded at a density of 2 ×
105 cells per well in an uncoated, 6 well tissue culture plate. 72 h
after plating, when cells were approximately 80% confluent, cells
were treated with either 0.1% DMSO or 1 ÎijM DC473 for 4 h,
then some samples were irradiated with UV-A (363-385 nm with
a peak at 371 nm; see ESI†) for 1 min. RNA was collected 1 h after
irradiation and prepared for sequencing using a Qiagen RNeasy
Mini Kit with on-column DNaseI digestion. RNA Samples were
quantified using the NanoDrop and fluorometric methods and the
mRNA had RIN values of 8 and above. RNAseq was performed
by the sequencing facility at the Institute for Genetic Medicine,
Newcastle University.
Illumina reads were aligned to the human reference
genome (GRCh38) using HISAT2 v.2.0.5.29.
ments were assembled and quantification performed using
StringTie v.1.3.4.30. The R package Ballgown v.2.6.0 was
The align-
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used for all differential expression analysis. Results from us-
ing the list of genes for differential expression (pval<0.01)
was compared by gene set enrichment analysis (GSEA;
http://software.broadinstitute.org/gsea/index.jsp) looking at the
‘GO’ (gene ontology) gene sets (see ESI†). These show the num-
ber of genes in each list which have a significant overlap with
particular sets of genes.
4 C. Schweitzer, Z. Mehrdad, A. Noll, E.-W. Grabner and
R. Schmidt, J. Phys. Chem. A, 2003, 107, 2192–2198.
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Science, 2010, 330, 517–521.
4.10 Cell death quantitation and statistical analysis
9 K. Ito, A. Hirao, F. Arai, S. Matsuoka, K. Takubo, I. Ham-
aguchi, K. Nomiyama, K. Hosokawa, K. Sakurada, N. Naka-
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Each well was allocated 4 set points where 3 points were sub-
jected to 1 min of UV irradiation in a specific rectangular zone.
The fourth zone was a control with no irradiation. Images were
captured at 30 min intervals for 24 h. Using ImageJ,66 time zero
and 24 h post irradiation numbers of viable and non-viable cells
were counted from the zone areas. The viable and non-viable cell
numbers for each time point were added together and the viabil-
ity percentage was calculated by dividing the viable cells by the
total cell count. The percentage of relative viability was then cal-
culated by dividing the 24 h time point by the zero time point.
Statistical significance was calculated by one-way ANOVA using
GraphPad Prism 7 software, Macintosh, GraphPad Software, La
Jolla California USA, www.graphpad.com.
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Molecules, 2011, 16, 4140–4164.
4.11 Zebrafish experiments
48 Hour, wild type zebrafish embryos (Golden) were treated with
1 µM DC324 diluted in E3 water (5.0 mM NaCl, 0.17 mM KCl,
0.33 mM CaCl2, 0.33 mM MgSO4) for 2 h.67 Embryos were then
transferred to fresh E3 water and incubated for a further 2 h prior
to irradiation. Embryos were anesthetised using buffered MS222
solution (0.1%; pH 7.0; ethyl 3-aminobenzoate methanesulfonate
dissolved in E3 water and buffered with NaHCO3) and then irra-
diated using the Axio Vert A1 system as described above with 33
J/cm2 of UV-A with appropriate environmental controls. Phase
contrast and fluorescence images were captured at the time of ir-
radiation with only phase contrast imaged captured at subsequent
time points.
16 A. Orenstein, G. Kostenich, L. Roitman, Y. Shechtman,
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18 V. B. Christie, J. H. Barnard, A. S. Batsanov, C. E. Bridgens,
E. B. Cartmell, J. C. Collings, D. J. Maltman, C. P. F. Red-
fern, T. B. Marder, S. Przyborski and A. Whiting, Org. Biomol.
Chem., 2008, 6, 3497–3507.
Conflicts of interest
A.W and C.A.A own shares of LightOx Limited, the company li-
censed to pursue commercial applications of the novel chemicals
described in this manuscript.
19 G. Zhou, D. M. Tams, T. B. Marder, R. Valentine, A. Whiting
and S. A. Przyborski, Org. Biomol. Chem., 2013, 11, 2323–
2334.
20 J. H. Barnard, J. C. Collings, A. Whiting, S. A. Przyborski and
T. B. Marder, Chem. Eur. J., 2009, 15, 11430–11442.
21 G. Clemens, K. R. Flower, P. Gardner, A. P. Henderson, J. P.
Knowles, T. B. Marder, A. Whiting and S. Przyborski, Mol.
BioSyst., 2013, 9, 3124–3134.
Acknowledgements
D.R.C. thanks the EPSRC, BBSRC and High Force Research Ltd.
for doctoral funding. T.B.M. thanks the Universität Würzburg for
support. A.K.N. thanks the Fulbright Foundation for a Visiting
Fulbright Scholarship. We thank Dr. R. M. Edkins for obtaining
some of the optical spectra of DC324. We thank Dr. E. Pohl for
helpful discussions and proof-reading.
22 G. Clemens, K. R. Flower, A. P. Henderson, A. Whiting,
S. A. Przyborski, M. Jimenez-Hernandez, F. Ball, P. Bassan,
G. Cinque and P. Gardner, Mol. BioSyst., 2013, 9, 677–692.
23 H. Haffez, D. R. Chisholm, R. Valentine, E. Pohl, C. P. F. Red-
fern and A. Whiting, Med. Chem. Commun., 2017, 8, 578–592.
24 H. Haffez, T. Khatib, P. McCaffery, S. Przyborski, C. Redfern
and A. Whiting, Mol. Neurobiol., 2018, 55, 1942–1950.
25 J. B. Bauer, W. P. Lippert, S. Dörrich, D. Tebbe, C. Burschka,
V. B. Christie, D. M. Tams, A. P. Henderson, B. A. Murray, T. B.
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