A multifunctional, light-activated prochelator inhibits UVA-induced oxidative stress

Article abstract of DOI:10.1016/j.bmcl.2015.06.048

UVA radiation can damage cells and tissues by direct photodamage of biomolecules as well as by initiating metal-catalyzed oxidative stress. In order to alleviate both concerns simultaneously, we synthesized a multifunctional prochelator PC-HAPI (2-((E)-1-

Full text of DOI:10.1016/j.bmcl.2015.06.048

Accepted Manuscript
A multifunctional, light-activated prochelator inhibits UVA-induced oxidative
stress
Andrew T. Franks, Qin Wang, Katherine J. Franz
PII:
S0960-894X(15)00641-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.048
BMCL 22838
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 May 2015
9 June 2015
11 June 2015
Please cite this article as: Franks, A.T., Wang, Q., Franz, K.J., A multifunctional, light-activated prochelator inhibits
UVA-induced oxidative stress, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/
j.bmcl.2015.06.048
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A multifunctional, light-activated
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Andrew T. Franks, Qin Wang and Katherine J. Franz*
2

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
A multifunctional, light-activated prochelator inhibits UVA-induced oxidative
stress
Andrew T. Franks, Qin Wang and Katherine J. Franz*
Duke University, Department of Chemistry, 124 Science Dr., Durham, NC 27708, USA.
* Tel: +1(919) 660 1541; E-mail address: katherine.franz@duke.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
UVA radiation can damage cells and tissues by direct photodamage of biomolecules as well as
by initiating metal-catalyzed oxidative stress. In order to alleviate both concerns simultaneously,
we synthesized a multifunctional prochelator PC-HAPI (2-((E)-1-(2-
Accepted
Available online
isonicotinoylhydrazono)ethyl)phenyl (trans)-3-(2,4-dihydroxyphenyl)acrylate) that contains a
trans-(o-hydroxy)cinnamate ester photocleavable protecting group that is cleaved upon UVA
exposure to release a coumarin, umbelliferone, and an aroylhydrazone metal chelator, HAPI
(N’-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide). While the prochelator PC-HAPI
exhibits negligible affinity for iron, it responds rapidly to UVA irradiation and converts to an
iron-binding chelator that inhibits iron-catalyzed formation of reactive oxygen species and
protects cells from UVA damage.
Keyword_1
Keyword_2
Keyword_3
Keyword_4
Keyword_5
Keywords: chelator; photocleavage; oxidative stress; UVA damage; antioxidant
2009 Elsevier Ltd. All rights reserved.
Radiation in the ultraviolet A (UVA) range (320–400 nm) of
the electromagnetic spectrum is a contributor to skin and retinal
tissue damage associated with aging.1-3 These longer UV
wavelengths pass through the atmosphere and penetrate more
deeply into biological tissues than the shorter UVB wavelengths
(290–320 nm). A number of biological chromophores absorb
UVA light, including enzyme cofactors (e.g. porphyrins and
flavins), pigments (eumelanin and pheomelanin) and other amino
acid derived species.4-6 In addition to dissipating the absorbed
energy through photon emission or radiationless decay, these
excited chromophores can propagate reactive excited states in
neighboring biomolecules via energy transfer mechanisms and
further stress cellular systems via reaction with molecular oxygen
to form singlet oxygen, superoxide and hydrogen peroxide.7-9
studies have shown, however, that oxidative damage induced by
both light and Fe can be alleviated by treatment with chelating
agents.10,16-21
While the clinical benefits of chelation therapy are clear for
remediation of some select diseases associated with Fe overload,
care must be taken to avoid the unintended consequences of
altering systemic metal balances.22 Basal levels of Fe are required
for cellular respiration and other crucial cellular processes;
therefore, systemic loss of Fe and long-term disruption of
homeostasis by chelating agents could also cause harm.23-25
A
prochelator strategy with responsive unmasking and selective
targeting of only unregulated and damaging species of Fe is
desirable to avoid possible side effects from systemic metal
depletion.26,27 Pourzand et al. introduced
a light-activated
UVA radiation is also known to cause cell damage via iron-
dependent mechanisms.10,11 Exposure of fibroblasts and
keratinocytes to environmentally relevant doses of UVA has been
shown to release iron (Fe) from cellular stores in both ferritin and
heme.12 While normal fluctuations in cellular Fe are moderated
by homeostatic machinery, the amount of reactive Fe that is
released from moderate to severe UVA exposure can overwhelm
this natural Fe buffering system. Iron that is not regulated
sufficiently can undergo Fenton chemistry and generate highly
reactive hydroxyl radicals as well as alkoxy and peroxy
radicals.13-15 These reactive oxygen species (ROS) can damage
DNA, proteins and lipids with diffusion-limited kinetics. Several
protecting moiety to mask the key metal-binding oxygen of two
extensively studied chelators, salicylaldehyde isonicotinoyl
hydrazone (SIH) and pyridoxal isonicotinoyl hydrazone (PIH) to
afford corresponding prochelators with negligible Fe affinity.28
Irradiation of the prochelators with UVA wavelengths effects an
intramolecular oxygen transfer within the (o-nitrobenzyl)ethyl
protecting group with a concomitant release of active SIH or
PIH. In this way, only labile Fe that is present near sites of UVA
exposure is targeted for chelation. However, while this strategy
has been suitable for biochemical studies in the lab, concerns
persist about possible cytotoxic effects from the nitrosoketone
byproduct released as a result of uncaging.29
1

In our search for an alternative uncaging strategy with
concomitant release of functional yet nontoxic components, we
reasoned that a trans-(o-hydroxy)cinnamate ester photocleavable
protecting group that releases a coumarin photoproduct would be
favorable.30-34 Coumarins are among several classes of abundant
and potent plant-based antioxidants.35,36 The diversity of
substituents around the heterocyclic core of natural coumarins
have inspired structure-activity studies to explore and compare
their antioxidant potency.37-41 Among the simplest structures of
plant coumarins is 7-hydroxycoumarin, also known as
umbelliferone, which is believed to be one of the active
ingredients in several medicinal plant extracts.42-44 Moreover, the
purported antioxidant activity of coumarins complements that of
antioxidant chelating agents. As sacrificial antioxidants,
coumarins mitigate oxidative stress by quenching oxygen-based
radicals stoichiometrically, while effective chelators prevent
redox-active metal ions from catalytically generating such
radicals in the first place. Since both direct UV damage of
biomolecules and catalytic production of hydroxyl radicals by Fe
contribute to UVA photodamage, a single molecule that could
mitigate both concerns may prove more effective than treating
either cause individually.
aqueous solution were irradiated under a 4 W handheld UV lamp
for 15 s intervals, which triggered a decrease in absorbance of
PC-HAPI at 284 and 350 nm with a concomitant increase of a
new feature at 325 nm. Three clean isosbestic points are
apparent, suggesting the photoconversion proceeds in one step
without side products or long-lived intermediates. The solution
was irradiated until no further changes were observed (60 s),
resulting in a final absorption spectrum that matches the
spectrum of a solution containing equimolar quantities of
authentic samples of umbelliferone and HAPI. Irradiation of
HAPI itself under these conditions also results in spectral
changes, as depicted by the subtle decreases in absorption in
Figure 1c. These changes can be attributed to partial E-Z
isomerization of the HAPI hydrazone bond, which we previously
reported.48 The intensity and duration of irradiation in the current
experiment is clearly not sufficient to fully photoisomerize the
HAPI sample, as the metastable Z isomer has very little
absorption at 400 nm.48 Interestingly, the spectrum of PC-HAPI
does not continue to lose absorbance at 400 nm upon longer
irradiation times, suggesting that the coumarin chromophore may
inhibit HAPI photoisomerization by a filter effect.
In the present report, a multifunctional prochelator has been
developed to address the simultaneous concerns of both Fe-
catalyzed and direct generation of ROS due to UVA exposure.
The compound PC-HAPI is designed using a “photocaged”
prochelator strategy. In this design, irradiation cleaves a
photolabile protecting group to yield the active form of a
chelator, in this case the lipophilic aroylhydrazone HAPI. An o-
hydroxycinnamic acid photoprotecting group has been chosen to
release the coumarin umbelliferone upon activation (Scheme 1).
Umbelliferone not only exhibits strong UVA absorption, but is
also reported to possess antioxidant properties.37,38,40,41,44
(a)
(b)
Scheme 1. Proposed deprotection mechanism for PC-HAPI
PC-HAPI was synthesized by following a synthetic scheme
modified from published o-hydroxycinnamate protections.30,45,46
Wittig chemistry was used to generate a trans-cinnamate ester.
Allyl ether protection of the cinnamate protecting group was used
to enable coupling with the HAPI phenolate while avoiding
unproductive reactions of the cinnamate with itself. Coupling of
the cinnamate protecting group to HAPI was done by in situ
generation of a cinnamoyl chloride and aryl ester formation in
pyridine solution (Figure S1). The product was obtained as a
pale yellow solid that dissolves readily in halogenated solvents
and is suitably soluble in DMSO for preparation of stock
solutions in the millimolar concentration range.
(c)
Photoinduced changes in the absorption spectrum of PC-HAPI
were monitored by UV-visible absorption spectrophotometry
(Figure 1). For comparison, authentic samples of HAPI and
ethyl (E)-3-(2,4-dihydroxyphenyl)acrylate (compound 1)47 were
also monitored to assess the independent response of the
aroylhydrazone framework and the cinnamate protecting group,
respectively, to UVA exposure. Samples in pH 7.4 buffered
Figure 1. Absorption spectra of (a) PC-HAPI, (b) compound 1, and (c) HAPI
in response to UVA irradiation. All solutions were prepared at 10 μM in pH
7.4 PBS buffer (<0.5% DMSO). Spectra prior to irradiation are shown as
thick, solid lines; intermediate spectra taken after 15 s intervals of irradiation
under a 4 W handheld lamp (maximum intensity at 366 nm) are shown as
gray lines; final spectra recorded after 60 s total UV exposure are drawn as
thick dashed lines. The final spectrum in the case of PC-HAPI (a) overlays
2

with that of a solution containing a mixture of 10 μM umbelliferone and 10
μM HAPI (black, solid line).
chelating species. To assess the metal binding behavior of PC-
HAPI before and after UV irradiation, the fluorescent metal
ligand calcein was used as a competitive chelator in a microplate
assay. Coordination of Fe3+ quenches the fluorescence of calcein,
so a competing chelator can restore calcein emission by binding
the metal and releasing free calcein. As seen in Figure 4,
quenched Fe(calcein) solutions treated with HAPI show a strong
return of fluorescence as Fe3+ is efficiently removed from its
complex with calcein. Treatment with the prochelator PC-HAPI
fails to restore emission, indicating that the Fe affinity of HAPI
has been attenuated by the photolabile masking group.
Treatment of Fe(calcein) with a solution of PC-HAPI that was
irradiated under UVA light prior to mixing results in near
complete restoration of calcein fluorescence. The UVA-
dependent increase in calcein fluorescence following treatment
with PC-HAPI indicates the release of an active chelator that is
able to compete with calcein for chelatable Fe.
Coumarins are recognized for their fluorescence properties, so
we anticipated observing an increase in fluorescence upon release
of umbelliferone during photodeprotection of PC-HAPI.
Emission spectra were collected for PC-HAPI before and after
incremental exposure to UVA radiation. As shown in Figure 2, a
pre-UVA solution is only weakly fluorescent, but after 10 s of
UVA exposure a strong growth in emission is observed from the
irradiated PC-HAPI solution.
No further increases were
observed after 60 s of UVA exposure. The final spectrum
collected exhibits a 4-fold increase in emission over the initial
spectrum and matches an umbelliferone standard of the same
concentration.
10
8
250
200
150
100
6
4
50
PC-HAPI
2
0
0
0
5
10
15
15
0
150
100
50
400
450
500
550
600
wavelength (nm)
Figure 2. Fluorescence emission of PC-HAPI increases upon exposure to
UVA radiation. PC-HAPI is weakly fluorescent in aqueous solution prior to
UVA exposure (black trace). Irradiation for 15, 30, 45, and 60 s (red trace)
with UVA causes an increase in fluorescence. [PC-HAPI] = 1 μM in PBS
(0.1% DMSO) λex = 360 nm. UVA irradiation was delivered by a 4W
handheld lamp with maximum intensity at 366 nm.
PC-HAPI + UV
0
5
10
150
100
50
umbelliferone
HAPI
In order to fully characterize the products of photoreaction,
the starting materials and products were analyzed by NMR
spectrometry (Figure S2) and liquid chromatography-mass
spectrometry (LC-MS) (Figure 3). Both methods show the clean
conversion of PC-HAPI to HAPI and umbelliferone. LC-MS
analysis of PC-HAPI solutions before and after UVA irradiation
shows the UV-induced decrease in [PC-HAPI] with concomitant
appearance of two new major peaks at 8.3 min and 11.6 min. The
elution times and corresponding mass spectra for these peaks
match authentic samples of umbelliferone and HAPI,
respectively. A minor species appearing earlier in the run has a
corresponding parent ion mass-to-charge ratio of 256, which is
also consistent with the molecular mass of HAPI. We assign this
peak as the (Z)-HAPI photoisomer.48 These results support the
proposed uncaging mechanism depicted in Scheme 1, wherein
absorption of UVA wavelengths of light induces cis-trans
isomerization of the cinnamate olefin. Isomerization allows for
intramolecular nucleophilic substitution that releases HAPI and
generates a fluorescent coumarin species.
0
0
5
10
15
elution time (minutes)
Figure 3. UV-triggered conversion of PC-HAPI to umbelliferone and HAPI
as monitored by LC-MS. (top) A solution of PC-HAPI kept in the dark elutes
as a single peak at 12.0 min. (middle) Irradiation of the solution with UVA
light (75 s in a photoreactor) generates two new eluting species (m/z = 163 at
8.3 min and m/z = 256 at 11.7 min). (bottom) Authentic samples of
umbelliferone (m/z = 163 for [M+H]+) and HAPI (m/z = 256 for [M+H]+)
elute at identical times as the products formed from irradiation of PC-HAPI.
All solutions were prepared at 50 μM in PBS (0.5% DMSO).
Given that PC-HAPI exhibits favorable prochelator properties,
we further tested its effectiveness for inhibiting Fe-dependent
formation of ROS. In the absence of suitably deactivating
ligands, hydrogen peroxide oxidizes Fe2+ to Fe3+ and generates
hydroxyl radical in aqueous solutions. If a reducing agent is also
present to recycle Fe3+ back to Fe2+, this reaction can be catalytic
in Fe, resulting in dramatically increased ROS production that
can be measured by the two-electron oxidation of non-fluorescent
dihydrodichlorofluorescein (H2DCF). The fluorescence emission
of the oxidation product dichlorofluorescein (DCF) can thus be
monitored to assess a compound’s affect on ROS production. If a
protective affect is observed at low compound concentrations, it
can indicate the presence of an Fe chelator that prevents catalytic
ROS formation, whereas compounds that diminish DCF
fluorescence at higher concentrations can indicate a capability of
The quantum yield of photolysis (ΦP) for the PC-HAPI
photoreaction was determined by using LC-MS. The extent of
PC-HAPI photolysis was compared under identical conditions to
that of compound 1, for which ΦP is known. The quantum yield
was found to be 8.1 ± 0.2%, which is slightly less than the value
determined for compound 1 (9%), which has the same protecting
group moiety but with an ethanol leaving group.47
A functional prochelator should have a low metal affinity until
acted on by the trigger stimulus, which releases a strong
3

quenching the ROS that forms. Solutions containing H2DCF were
incubated with Fe3+, hydrogen peroxide, ascorbic acid and
nitrilotriacetic acid (NTA, used to keep Fe in solution). As shown
by the bar on the far right in Figure 5, these conditions generate
significant DCF fluorescence. Treated wells were dosed with
HAPI or PC-HAPI with (+) or without (-) UVA irradiation.
Emission levels from wells containing only H2DCF and buffer
were stable over the time period of the experiment, and this
background emission was subtracted from readings for all control
and treatment wells.
Fe-catalyzed ROS production, but may provide antioxidant
properties of ROS quenching at high concentrations. Emission
drops markedly in wells treated with irradiated PC-HAPI
compared to wells containing the same concentration of non-
irradiated PC-HAPI. The light-dependent DCF emission levels
suggest that PC-HAPI is unable to prevent Fe redox cycling until
it is activated by UVA radiation. The photolyzed sample, on the
other hand, shows inhibition of Fe redox catalysis at low to
moderate concentrations. However, the 100 μM and 200 μM
photolyzed PC-HAPI samples still exhibit some DCF
fluorescence and therefore do not fully replicate the behavior
observed with corresponding HAPI solutions, which show
negligible DCF emission.
In order to further probe why irradiated samples of PC-HAPI
do not fully prevent DCF fluorescence, the other PC-HAPI
photoproduct, umbelliferone, was also examined by this assay
(Figure 6). H2DCF solutions containing the same ROS-
generating reagents were treated with a range of umbelliferone
concentrations and, for comparison, the water-soluble antioxidant
trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid). Whereas trolox effectively inhibits DCF oxidation under
these conditions, umbelliferone shows a dose-dependent increase
in DCF emission. Because of the very different excitation and
emission profiles of umbelliferone and DCF, this growth in
emission is not due to umbelliferone fluorescence itself. This
result suggests that umbelliferone promotes, rather than
quenches, ROS production under these Fenton-like conditions.
While this behavior is unexpected and is incongruous with the
reported antioxidant behavior of coumarin phenols, it is only
observed at much higher concentrations than would be
administered in a biological application.
Figure 4. A comparison of calcein fluorescence emission intensity shows that
the Fe3+ affinity of PC-HAPI (PC) is controlled by light. [Calcein] = [Fe] = 2
μM, where Fe = [(NH4)2Fe(SO4)2], which forms Fe3+(calcein) complex in situ;
[HAPI] = [PC-HAPI] = 10 μM in PBS. Emission recorded 1 h after
photolysis (+) for 15 s in a photoreactor (λmax = 350 nm). Error bars represent
standard deviation for conditions run in triplicate.
DCF emission of solutions treated with HAPI decreased in a
dose-dependent manner (Figure 5). This behavior is consistent
with the protective effects observed in previous studies of HAPI
and related aroylhydrazone chelators, where sequestration of the
active metal center inhibits ROS production.49-52 Wells treated
with non-irradiated PC-HAPI show much higher levels of DCF
emission, with a decrease observed only at the highest 200 M
dose. This result indicates that PC-HAPI itself does not prevent
To investigate the potency of PC-HAPI to protect cells
subjected to UVA damage, retinal pigment epithelial ARPE-19
cells were chosen as they suffer from UVA-induced oxidative
stress during age-related macular degeneration (AMD).53 As
indicated in Figure 7, cells exposed to UVA irradiation were
protected from cell death by both HAPI and PC-HAPI, while
umbelliferone itself provided little protection. HAPI was
protective at all concentrations tested from 5 to 100 M, while
Figure 5. Fluorescence assay for Fe-mediated ROS production. HAPI,
PC-HAPI, and photolyzed PC-HAPI were tested for their ability to
prevent oxidation of H2DCF (20 μM) to fluorescent DCF by a cocktail
of 10 μM FeCl3, 10 μM NTA, 1 mM ascorbic acid, and 1 mM H2O2 in
pH 7.4 PBS buffer. All solutions contained 10 μM NTA as a
solubilizing Fe3+ ligand. (“NTA” condition contains NTA with no
additional chelators). ex = 485 nm, em = 535 nm
Figure 6. Fluorescence assay of the effect of umbelliferone and trolox on
Fe-mediated ROS production monitored by DCF emission. All solutions
were prepared in PBS (pH 7.4) with 10 μM FeCl3, 10 μM NTA, 1 mM
H2O2 and 1 mM ascorbate. Condition labeled “NTA” contains only Fe,
NTA, H2O2, and ascorbate.
4

PC-HAPI showed greatest protection against UV damage at
doses lower than 50 μM, with less protection at the higher doses.
Cytotoxicity studies revealed that PC-HAPI, HAPI, and
umbelliferone were nontoxic at concentrations up to 100 M
after 24 h incubation. ARPE-19 cells were less tolerant of high
doses of HAPI and PC-HAPI when incubation was extended over
72 h, although doses below 25 M showed minimal toxicity even
after 72 h.
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In summary, we seek to improve upon previous efforts to
sequester reactive Fe using light-triggered prochelators by
releasing not only one but two functional photoproducts with
useful properties for attenuating UV photodamage. The newly
synthesized compound PC-HAPI responds sensitively to UVA
exposure, releasing both the coumarin umbelliferone and the
aroylhydrazone metal chelator HAPI. The prochelator PC-HAPI
has weakened affinity for Fe due to the acylation of the metal-
binding phenolate oxygen. Upon exposure to UVA radiation, the
free phenol is released and restores the affinity for Fe. Assays of
Fe-catalyzed ROS generation using dihydrodichlorofluorescein
indicate that the released chelator HAPI attenuates ROS
generation, while umbelliferone may act as a pro-oxidant at high
concentrations. Cell studies further demonstrate that PC-HAPI
and its corresponding chelator HAPI effectively protect retinal
pigment epithelial cells from UVA irradiation. In contrast, the
insignificant cytoprotective effect of umbelliferone is likely to
derive from its pro-oxidant potential. The activity of several other
naturally occurring and synthetic coumarins have identified
numerous compounds with highly potent antioxidant and anti-
inflammatory activity. Future iterations of cinnamate-protected
prochelators could be designed to release these potent coumarins
and thus confer greater protective effects against harmful UV
damage.
41. Paya, M.; Halliwell, B.; Hoult, J. R. S. Biochem. Pharmacol. 1992, 44,
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Acknowledgement. We thank the National Institutes of Health
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Supplementary Material
Supplementary material is available for download and
includes all experimental details and supporting data figures
mentioned in the main text.
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