A prochelator with a modular masking group featuring hydrogen peroxide activation with concurrent fluorescent reporting

Article abstract of DOI:10.1039/c4cc05076b

Metal chelators masked with protecting groups for targeted release have the potential to conditionally modulate cellular metals. We report a new route to prepare cis-cinnamate protecting groups that enabled development of a prochelator with chemical stimulus response, fluorescent reporting and active compound release in a single structure. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc05076b

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A prochelator with a modular masking group
featuring hydrogen peroxide activation with
concurrent fluorescent reporting†
Cite this: Chem. Commun., 2014,
50, 11317
Received 3rd July 2014,
Accepted 5th August 2014
Andrew T. Franks and Katherine J. Franz*
DOI: 10.1039/c4cc05076b
www.rsc.org/chemcomm
Metal chelators masked with protecting groups for targeted release
have the potential to conditionally modulate cellular metals. We
report a new route to prepare cis-cinnamate protecting groups that
enabled development of a prochelator with chemical stimulus
response, fluorescent reporting and active compound release in a
single structure.
Misregulation of biological metal ions has been implicated as a
contributor to many diseases. Altering the concentration and
distribution of select metal ions can therefore be a powerful
pharmacological strategy for fighting disease. For example,
b-thalassemia patients are often treated with the chelating agent
desferrioxamine to avoid organ damage from iron overload.1 In
Wilson’s disease, chelation therapy can ameliorate neurological
and hepatological symptoms associated with malfunctioning
Scheme 1 Activation of the fluorogenic prochelator BCQ with hydrogen
peroxide.
copper transport.2 However, administering therapeutic chelators lactonization reaction was previously utilized as a deprotection
carries unintended risks of systemic metal depletion and associated strategy for esterase-sensitive prodrugs.5 The concept was
toxic effects.1,3 Our laboratory and others therefore seek to also notably extended to benzylidenemalonates for triggerable
develop molecules, termed ‘prochelators’, that bind metals only luminescence sensing.6 As shown in Scheme 1, BCQ relies on
under disease conditions. We recently reported that a non-toxic oxidation of an aryl boronic ester to yield an unstable phenol
prochelator named QBP converts to 8-hydroxyquinoline (8HQ) in that undergoes spontaneous 1,6-benzyl elimination to release
response to the oxidative burst of activated macrophages, an quinone methide (a reactive species that is ultimately quenched
event that releases reactive oxygen and nitrogen species that in water to 4-hydroxybenzyl alcohol, 3b) and a cis-(o-hydroxy)-
deprotect the boronate masking group.4 The released 8HQ elicits cinnamic acid ester. The exposed hydroxyl group is then expected
copper-dependent killing of a fungal pathogen in vitro and to attack intramolecularly the electrophilic carboxylate carbon, with
in mouse pulmonary infection.4 A multifunctional agent that collapse of the tetrahedral intermediate leading to ejection of the
provides a fluorescent signal upon activation would be useful to 8HQ metal-binding unit7 simultaneously with 7-hydroxycoumarin
probe the localization and timing of such agents in order to under- (umbelliferone, Umb). Umbelliferone is a well-known fluorophore,
stand their mechanism of action and improve disease targeting.
absorbing near-UV and emitting in the blue region of the visible
The present work describes the preparation of such a multi- spectrum. By releasing equimolar quantities of chelator and
functional prochelator, named BCQ. The design takes advantage of fluorophore, real-time monitoring of prochelator activation
the intramolecular acyl substitution of cis-(ortho-hydroxy)cinnamic may be possible.
acid derivatives to form coumarin chromophores. The rapid
Reported syntheses of compounds with cis-coumaric acid
masking groups have primarily relied on reduction of the
coumarin carboxylate moiety or photochemical isomerization
of the trans-coumarate.8 Neither of these routes yielded sufficient
quantities of the cis configuration for our purposes. Other reported
reactions utilize conditions that would degrade the bromomethyl
Department of Chemistry, Duke University, Durham, NC, 27708, USA.
E-mail: Katherine.franz@duke.edu
† Electronic supplementary information (ESI) available: Synthetic protocol, com-
pound characterization data. See DOI: 10.1039/c4cc05076b
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Fig. 1 (a) A BCQ solution monitored by LC-MS 10 min following addition
of 5 mM H2O2. UV detection at 254 nm (blue trace) and 330 nm (red trace)
reveals only a trace signal of BCQ eluting B12 min, with new species
consistent with products 8HQ and Umb, as well as an intermediate (m/z =
414) and a side-product (m/z = 308). (b) Reference chromatograms of
unreacted BCQ and authentic 8HQ and Umb samples (all 50 mM in 1 : 1
PBS : MeOH + 2% DMSO).
Scheme 2 Synthesis of BCQ.
arylboronic ester reagent required for BCQ and were thus not
suitable.9 Instead, we developed an alternative synthetic route
for opening coumarin rings and installing a trigger that renders
the compound sensitive to H2O2.
corresponding m/z values of 308 and 414, respectively. The 414
BCQ was prepared in five steps starting with the deconstruction species, which is consistent with intermediate 1 in Scheme 1, is
of commercially available Umb (Scheme 2). Because this structure not observed in chromatograms collected 1 h after peroxide
will ultimately be reconstructed upon peroxide activation, the addition when the starting material has been B99% consumed
scheme allows the flexibility to incorporate alternative coumarin (data not shown). This result fits a deprotection mechanism
derivatives that could be desirable for their different biological or wherein rapid oxidation of the boronate moiety precedes rate-
physical properties.
limiting 1,6-benzyl elimination. The 4-hydroxybenzyl alcohol
Allyl protection of the coumarin is suitable to block its was not observed by LC-MS, but its presence as a reaction
phenol during subsequent esterification with 8HQ and is stable product was verified by NMR (d (ppm): 7.28 (d, J = 6 Hz, 2H);
to both acidic and basic conditions used in the synthesis. The 6.79 (d, J = 6 Hz, 2H)).10 These results support the proposed
opening of the lactone ring in 4 under strongly basic conditions activity of BCQ as a peroxide-dependent source of 8HQ and Umb.
enables alkylation of the exposed phenolate with the bromomethyl
The small signal with m/z = 308 observed after 10 min of
arylboronic ester, thereby preventing ring closure upon quenching. reaction persists with the same relative abundance even 12 h
Coupling with 8HQ, final deprotection, and purification by column after peroxide addition (data not shown). This mass fits that
chromatography yields the product as one isomer, which by expected for a species with molecular formula C18H14NO4 ([M + H]+
1H NMR spectroscopy reveals a 3J H–H coupling constant of ion), consistent with intermediate 2 in Scheme 1.
13 Hz that is characteristic for cis olefins. BCQ is obtained as a
white solid dissolvable in DMSO in high concentrations useful by spectroscopic evidence. The fluorescence emission of a BCQ
as stock solutions. solution was monitored following treatment with a 100-fold
The proposed BCQ activation scheme is further supported
The reaction of BCQ with H2O2 was monitored by liquid molar excess of H2O2 (Fig. 2). Upon mixing, the solution
chromatography-mass spectrometry (LC-MS) (Fig. 1). Prior to displayed a time-dependent growth of emission intensity. The
peroxide addition, BCQ elutes as the boronic acid species at resulting spectra (lex = 350 nm, lem,max = 454 nm) are consistent
11 min (m/z = 442). The lability of the pinacol ester in HPLC with Umb fluorescence. After reaction completion, the integrated
conditions has been previously reported.10 After 10 min of reaction, emission intensity is 90-fold greater than that of the starting
very little BCQ remains, but new peaks appear at 2.0 and 8.6 min solution. The emission data fit a first-order reaction model with
with respective mass-to-charge (m/z) signals of 146 and 163 and an observed pseudo-first order rate constant kobs = 0.010 sÀ1
absorption profiles best visualized at 254 and 330 nm, respectively (Fig. 2 inset).
(Fig. 1a). These data match authentic 8HQ and Umb samples run
The absorption spectrum of BCQ in 50 : 50 PBS : methanol
under identical conditions (Fig. 1b). In addition to these prominent shows three peaks at 301, 314, and 335 nm (Fig. 3). Due to the
peaks, two smaller peaks elute at 10.5 and at 11.5 min with cinnamate chromophore, BCQ has significant absorption
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Fig. 4 Treatment of BCQ with 200 (K), 25 (E), 10 (m), or 0 mM H2O2 on a
microplate results in dose-dependent fluorescence increases. Samples
were incubated at 25 1C (blue) or 37 1C (red). Error bars for 200 mM samples
represent the standard deviation from triplicate samples and are repre-
sentative for the entire set. BCQ solutions were diluted to 2 mM in PBS from
1 mM stock in DMSO.
Fig. 2 A 1 mM solution of BCQ in PBS (0.1% DMSO) is nonfluorescent.
Addition of 100 mM H2O2 results in growth of emission over time (lex
=
350 nm).
extending to B425 nm. As the reaction with peroxide proceeds,
the spectra shift with formation of isosbestic points at 305 and
410 nm. The spectrum obtained at 90 min (after no further
spectral change is observed) bears the same features as a
solution containing equimolar 8HQ and Umb.
Kinetic analysis of the UV-visible data collected under
pseudo-first order conditions gives a second order rate constant
of 0.54 MÀ1 sÀ1 for the appearance of product. This rate constant is
consistent with those observed for other compounds protected with
benzyl ether-linked boronic esters.10,11 We conclude from this
observation that the inclusion of the pro-coumarin cinnamate
moiety does not alter 8HQ release kinetics in this system.
was then monitored over several hours. The experiment was
performed both at 25 1C and 37 1C (Fig. 4). Wells containing as
little as 2.5 mM H2O2 elicited discernible increases in fluorescence
compared to negative control wells. However, the observed emis-
sion from activated BCQ wells did not match the intensities
observed in wells loaded with Umb at the same concentration.
While spectroscopic and chromatographic data provide
evidence of 8HQ and Umb release, results from these experi-
ments consistently indicate that less product is generated than
would be predicted based on the initial concentration of BCQ. A
possible explanation for this deficit is a thermal isomerization
of the cis configuration of intermediate 2 to the trans configu-
ration. Such a change would reposition the ester and thereby
disfavor lactonization. Only one unexpected species was
observed in the final H2O2 reaction mixture: a compound that
elutes at 10.5 min with an m/z value of 308 in the LC-MS
(Fig. 1a), which is consistent with either a cis or trans species 2.
The cis-(o-hydroxy)cinnamate should undergo rapid cyclization
to produce the product coumarin. This process clearly occurs
on a rapid timescale given the appearance of coumarin in the
chromatograms. The persistence of the m/z 308 species hours
after reaction completion is consistent with the presence of a
deactivated intermediate 2.
To gauge its feasibility for use in biological assays, BCQ (2 mM in
PBS buffer) was mixed in microplate wells with H2O2 ranging in
concentration from 156 nM to 200 mM. Umbelliferone emission
The temperature-dependent differences in emission inten-
sity observed in the microwell plate assay also support this
hypothesis (Fig. 4). Wells incubated at 37 1C during activation
exhibited substantially lower emission than those at room
temperature; the increase in thermal energy would speed up
not only peroxide activation but also the undesired isomeriza-
tion reaction. Based on these combined data, we attribute the
unexpected mass and the temperature dependence of emission
to a byproduct trans-2.
BCQ exhibits many desirable attributes for a chemical tool
to modify local metal coordination environments in response
to an oxidative stimulus. Fluorescence detection could be used
in microplate assays or fluorescence microscopy to learn more
about the interaction of prochelators with oxidatively stressed
cells. However, there are several characteristics of BCQ that
Fig. 3 Absorption spectra show the change in spectral features as BCQ
(dashed line, 20 mM in 1 : 1 PBS : MeOH) converts to products over the course
of 90 min following addition of 1 mM H2O2 (t = 90 min, black, bold line).
Intermediate spectra shown were collected at 5 min intervals. A solution of
20 mM 8HQ + 20 mM Umb in 1 : 1 MeOH : PBS (red, bold line) shown for
comparison. Left inset: absorbance vs. time data fit a pseudo-first order model
for product formation during the reaction (red line). Right inset: a linear fit of
kobs vs. [H2O2] gives the second-order rate constant for product formation.
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warrant further development. BCQ exhibits low solubility in Fund administered by Duke Chemistry for fellowship support
aqueous solutions without organic co-solvent, limiting its use to ATF.
to low concentrations (o5 mM) or organic–aqueous solvent
mixtures. Although Umb fluorescence is detectable at these
low concentrations, studies from our laboratory suggest that
Notes and references
higher prochelator concentrations are required to alter the fate
of stressed cells.4,10 Consequently, improvements are desirable
for future applications in living cells.
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The turn-on response of BCQ could be enhanced by mini-
mizing the thermal deactivation of the cis isomer. Depletion of
the active form by isomerization lowers the yield of released
chelator and fluorophore, so efforts to inhibit this process
would benefit both activity and sensitivity. The reactivity of
BCQ was explored here with H2O2, though it is important to
keep in consideration that aryl boronates also undergo facile
deprotection by peroxynitrite (ONOOÀ).12
In conclusion, a new synthetic route to transform coumarin
fluorophores into stable cis-cinnamates enabled the development
of a stimulus-responsive prochelator with fluorescent reporting. The
synthesized compound, BCQ, demonstrates reactivity to hydrogen
peroxide that results in oxidation of a boronic ester, followed by
spontaneous 1,6-benzyl elimination and intramolecular nucleophilic
substitution to release a desired metal chelator and an emissive
fluorophore. The unactivated prochelator is nonfluorescent and
exhibits a 90-fold increase in emission intensity upon exposure to
hydrogen peroxide. BCQ signifies a proof of principle and first
generation attempt to incorporate stimulus response, visual readout, 11 (a) J. L. M. Jourden, K. B. Daniel and S. M. Cohen, Chem. Commun.,
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12 J. Zielonka, A. Sikora, M. Hardy, J. Joseph, B. P. Dranka and
and active compound release in a single molecule.
We thank Dr George Dubay for assistance with LC-MS, the
NIH for funding (GM084176), and the Burroughs Wellcome
B. Kalyanaraman, Chem. Res. Toxicol., 2012, 25, 1793.
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