Methyl 5-MeO-: N -aminoanthranilate, a minimalist fluorogenic probe for sensing cellular aldehydic load

Article abstract of DOI:10.1039/c8ob02255k

Methyl 5-MeO-N-aminoanthranilate, a fluorogenic probe comprising a single substituted benzene ring has been applied towards the fluorescence detection of endogenous carbonyls through rapid, catalyst-free complexation of these bio-derived markers of cell stress under physiological conditions. The products formed during the reaction between the probe and aldehydic products of lipid peroxidation, including malondialdehyde and long-chain aliphatic aldehydes relevant to the oxidative decomposition of cell membranes, have been evaluated. Live cell imaging of diethyl maleate-induced oxidative stress with or without pretreatment with α-tocopherol was carried out, with the result suggesting that the presented molecule might serve as a minimalist molecular probe capable of cellular "Aldehydic Load" detection by fluorescence microscopy. This work also outlines functional constraints of the fluorogenic probe (i.e. intramolecular cyclization), providing a realistic evaluation of methyl 5-MeO-N-aminoanthranilate for fluorescence-based aldehyde detection.

Full text of DOI:10.1039/c8ob02255k

Organic &
Biomolecular Chemistry
View Article Online
View Journal
PAPER
Methyl 5-MeO-N-aminoanthranilate, a minimalist
fluorogenic probe for sensing cellular aldehydic
load†
Cite this: DOI: 10.1039/c8ob02255k
Mojmír Suchý,a,c Caitlin Lazurko,a Alexia Kirby,b,c Trina Dang,a George Liua and
a,c
Adam J. Shuhendler
*
Methyl 5-MeO-N-aminoanthranilate, a fluorogenic probe comprising a single substituted benzene ring
has been applied towards the fluorescence detection of endogenous carbonyls through rapid, catalyst-
free complexation of these bio-derived markers of cell stress under physiological conditions. The pro-
ducts formed during the reaction between the probe and aldehydic products of lipid peroxidation, includ-
ing malondialdehyde and long-chain aliphatic aldehydes relevant to the oxidative decomposition of cell
membranes, have been evaluated. Live cell imaging of diethyl maleate-induced oxidative stress with or
without pretreatment with α-tocopherol was carried out, with the result suggesting that the presented
molecule might serve as a minimalist molecular probe capable of cellular “Aldehydic Load” detection by
fluorescence microscopy. This work also outlines functional constraints of the fluorogenic probe
(i.e. intramolecular cyclization), providing a realistic evaluation of methyl 5-MeO-N-aminoanthranilate for
fluorescence-based aldehyde detection.
Received 12th September 2018,
Accepted 1st November 2018
DOI: 10.1039/c8ob02255k
rsc.li/obc
denoted herein as carbonyls) to form hydrazones.4 When
anthranilic acid is converted into the corresponding hydrazine
Introduction
Anthranilic acid (1a, Fig. 1) and methyl anthranilate (1b, (N-aminoanthranilic acid, 1c, Fig. 1), the rate of the reaction
Fig. 1) are among the simplest naturally occurring fluoro- with carbonyls increases dramatically even surpassing that
phores, exhibiting long excited state life times (8.7 ns) along associated with strain-promoted “click” reactions, which
with high quantum yields in aqueous solution (Φ ∼0.60).1 makes N-aminoanthranilates attractive reactive head-groups
Although these properties appear to make the anthranilic acid- for the rapid, catalyst-free trapping of biogenic carbonyls
based fluorophores attractive probes for molecular biology, under physiological conditions.5
their use in the context of the development of imaging probes
It is now well established that the exposure of humans to
is still in its infancy.2 This can be in part attributed to the fact carbonyls has a negative impact on health and well-being.6
that anthranilic acid-based fluorophores require high energy
excitation (320–350 nm) and emit in the violet to blue region
(λem 410–470 nm).1 Moreover, the synthetic methodologies
describing their incorporation into larger molecular entities
are rather limited.3
Kool and co-workers have recently shown that substituted
anthranilic acids can auto-catalyze the reaction between hydra-
zines and carbonyl compounds (aldehydes and ketones
aDepartment of Chemistry & Biomolecular Scences, University of Ottawa, Ottawa,
ON, Canada. E-mail: ashuhend@uottawa.ca
bDepartment of Biology, University of Ottawa, Ottawa, ON, Canada
cUniversity of Ottawa Heart Institute, Ottawa, ON, Canada
†Electronic supplementary information (ESI) available: Full spectral characteriz-
ation of hydrazones 3f–3i, fluorescence spectra associated with pyrazoles 4a and
4b, full view of the 1H NMR spectra associated with the detailed kinetic analysis.
Fig. 1 Structures of anthranilic acids and anthranilates 1a–1c, 2a, 2b,
hydrazones 3a–3i, pyrazoles 4a and 4b.
See DOI: 10.1039/c8ob02255k
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Although some carbonyls are present in the environment as a
consequence of industrial operations (e.g. acetone, formal-
dehyde), many simple carbonyls (e.g. acetaldehyde, glyceralde-
hyde, pyruvate) are also produced endogenously via tightly
Results and discussion
Preparation of hydrazone/pyrazole standards and evaluation of
their fluorescence properties
regulated metabolic pathways.6 Increased production of carbo- Chemical synthesis of the probes was carried out as outlined
nyls upon the disruption of homeostasis often times underlies in Scheme 1. Reaction of methyl 5-MeO-N-aminoanthranilate
early stages of various pathologies, including diabetes (e.g. (2b) with simple longer chain aliphatic aldehydes (pentanal,
acetone),7 cancer (e.g. simple aliphatic aldehydes)8 cardio- hexanal, octanal, nonanal) afforded hydrazones 3f–3i in good
vascular diseases [e.g. acrolein, malondialdehyde (MDA)]9 or yields (61–83%). The final compounds were purified by flash
traumatic brain injury (e.g. 3-aminopropanal).10 The ability to column chromatography (FCC) and were obtained as insepar-
properly design, synthesize and evaluate molecular probes able mixtures of E and Z isomers in approximately a 2 : 3 ratio
capable of in vitro or in vivo mapping of total endogenous (major isomer not identified). Observed results are in contrast
carbonyls (denoted herein as Aldehydic Load) is of significant with the synthesis of previously described hydrazones (3a–3e,
interest to the scientific community,11 as the outcomes Fig. 1),12 which were obtained either as a single isomer, or
of these research efforts are expected to facilitate our funda- where the amount of the minor isomer was negligible (<5%).
mental understanding of, and ability to diagnose, a wide Fluorescence spectra of hydrazones 3f–3i were acquired in
range of illnesses affecting a significant part of the global phosphate buffered saline (1× PBS, pH 7.2) containing up to
population.
The development of molecular imaging probes capable of determined using harmane as standard (Table 1).13
reporting on Aldehydic Load at both the in vitro and in vivo While the excitation wavelengths appear to be independent
10% DMSO (Fig. 2), and relative quantum yields (Φ) were
scales is one of the key research avenues currently pursued in of the hydrazone structure and only vary by 6 nm, the emission
our laboratory. We have recently shown that 5-MeO-N-ami- wavelengths as well as Stoke’s shifts decrease slightly with
noanthranilic acid (2a, Fig. 1), a simple anthranilic acid-based increasing length of the carbon chain. Fluorescence quantum
hydrazine can act as a Chemical Exchange Saturation Transfer yields were found to be relatively low, possibly a consequence
Magnetic Resonance Imaging (CEST MRI) contrast agent upon of the presence of two inseparable isomers, and decreased
reaction with endogenous aldehydes (e.g. acetaldehyde, 3-ami- with an increased length of aldehyde carbon chain. When
nopropanal).5b During the course of our studies, we have also 5-MeO-N-aminoanthranilic acid (2a) or methyl 5-MeO-N-ami-
prepared methyl 5-MeO-N-aminoanthranilate (2b, Fig. 1) and noanthranilate (2b) were treated with MDA tetramethyl acetal,
allowed it to react with several endogenous carbonyls. The pyrazoles 4a and 4b were obtained in moderate yield
majority of the products we obtained upon reaction with alde- (Scheme 1) upon purification by FCC as described recently.12
hydes (hydrazones 3a–3e, Fig. 1) were found to fluoresce in the
blue to blue-green region of the spectrum; therefore we
decided to study their fluorescence in greater detail. As
described recently, we have acquired excitation–emission
matrices (EEM) associated with compounds 3a–3e (Fig. 1), and
have concurrently developed a novel pattern matching algor-
ithm for the fluorescent fingerprinting of biogenic carbonyls.12
This approach allowed us to determine both the total amount
of aldehyde (i.e. Aldehydic Load), as well as the identity of indi-
vidual components of carbonyl mixtures. As an expansion to
our previous studies, we are describing herein the fluorescence
properties associated with hydrazones derived from longer
Scheme 1 Preparation of hydrazones 3f–3i and pyrazoles 4a and 4b.
chain aliphatic aldehydes as putative end-products of lipid per-
oxidation (compounds 3f–3i, Fig. 1), and validating the use of
2b as a fluorogenic probe capable of mapping Aldehydic Load
via live cell microscopy in a model of oxidative stress. We have
also carried out detailed mechanistic studies to elucidate the
mechanism of the reaction between methyl 5-MeO-N-aminoan-
thranilate (2b, Fig. 1) and the dialdehyde MDA to form a pyra-
zole ring through an in situ detected hydrazone intermediate.
The further understanding of the reaction mechanisms of
methyl 5-MeO-N-aminoanthranilate with biogenic carbonyls
reported in this work supports the utility of this minimalist
fluorogenic probe, and advocates for its value as a novel tool
Fig. 2 Emission scans for hydrazones derived from 2b and aliphatic
for studying the chemical biology of lipid peroxidation in live
cells.
aldehydes 3f (green), 3g (cyan), 3h (blue), and 3i (purple) acquired with
λex = 370 nm.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Organic & Biomolecular Chemistry
Paper
Table 1 Fluorescence properties associated with hydrazones 3f–3i
The development of a molecular imaging probe capable of
in situ complexation of MDA in live cells in order to more accu-
rately map concentrations has been challenging due to the
high reactivity of MDA in living systems, which is partly
attributable to its presence in two tautomeric forms.21 Recent
efforts toward this end include two green fluorescent probes
(8a,22 9a,23 Scheme 2), bearing a hydrazide/hydrazine subunit
reported to quench fluorescence via photoinduced electron
transfer (PET). Upon reaction with MDA, a five-membered pyr-
azolidine/pyrazole ring is formed (compounds 8b, 9b,
Scheme 2), limiting PET to result in a strong fluorescence
turn-on.22,23 Both 8a and 9a show excellent selectivity toward
MDA, as no fluorescence response is observed upon exposure
to other carbonyls.22,23 Interestingly, this observation is in con-
trast with our results using methyl 5-MeO-N-aminoanthrani-
late (2b), wherein the reaction with a range of simple endogen-
ous carbonyls leads to the formation of fluorescent hydrazones
(e.g. compounds 3a–3i).12 Although the detailed mechanism of
the fluorescence signal turn on associated with the 8a → 8b
transformation has been proposed based on NOESY measure-
ments,22 no evidence for the suggested mechanism associated
with the signal turn on upon 9a → 9b conversion has been pro-
vided.23 Due to differential aldehyde selectivity of 2b relative to
8a and 9a, a detailed mechanistic evaluation of the reaction of
MDA with 5-MeO-N-aminoanthranilic acid (2a) and methyl
5-MeO-N-aminoanthranilate (2b) was undertaken.
ϕ
λex
(nm)
ε
λem
(nm)
Stoke’s
shift (nm)
Compound
(harmane)
(m2 mol−1
)
3f
0.17
0.08
0.08
0.06
372
376
377
378
5631
7107
8410
8025
480
475
467
466
108
99
90
3g
3h
3i
88
Pyrazole formation from MDA and methyl 5-MeO-N-
aminoanthranilate (2b) proceeds through a long-lived
fluorescent hydrazone intermediate
Malondialdehyde (MDA, 5, Scheme 2), a terminal product of
polyunsaturated fatty acid peroxidation mediated by reactive
oxygen species (ROS) is regarded as a classical marker of oxi-
dative stress.14 MDA occurs as an equilibrium of two tauto-
mers (Scheme 2), is a known mutagen,15 and has been associ-
ated with early stages of various pathologies (e.g. cancer,16
atherosclerosis,17 stroke,18 Alzheimer’s disease19). MDA has
classically been assayed in cells or tissue extracts through treat-
ment with thiobarbituric acid (6, Scheme 2) with the formation
of a red coloured dye 7 under harsh conditions.20
Compound 2b (Fig. 1, Scheme 2) contains a hydrazine
subunit and is known to form a pyrazole ring (compound 4b,
Scheme 2) upon exposure to MDA.12 The structure of pyrazole
4b is simple, with well-resolved resonances in its 1H NMR
1
spectrum,12 which permitted a detailed H NMR-based kinetic
analysis to be carried out in order to fully understand the
transformation of 5-MeO-N-aminoanthranilic acid (2a) and
methyl 5-MeO-N-aminoanthranilate (2b) to pyrazoles 4a and
4b upon reaction with MDA.
Since the main chemical shift differences due to pyrazole
formation were expected in the aromatic region (6–8 ppm),
only changes occurring within this range are depicted in Fig. 3
and discussed below. Note that full views of the spectra can be
found in Supporting Fig. S1 and S2.† Briefly, within this
region both hydrazines 2a and 2b exhibit three distinct signals
(2a, d, 7.26 ppm, J = 3 Hz, H6; d, 7.08 ppm, J = 9 Hz, H3; dd,
7.01 ppm, J = 9, 3 Hz, H4; 2b, d, 7.52 ppm, J = 3 Hz, H6; dd,
7.20 ppm, J = 9, 3 Hz, H4; d, 7.05 ppm, J = 9 Hz, H3) fully con-
sistent with the substitution pattern present in 2a and 2b.
Baseline 1H-NMR spectra were acquired of separate solu-
tions of 1 mg of 2a or 2b in 1× PBS, pH 7.2 containing 10%
DMSO-D6, followed by the addition of 1 drop of a 1 M solution
of MDA tetramethyl acetal in 1 M HCl, known to form MDA
in situ.24 Further acquisitions of 1H NMR spectra occurred
immediately (1 min), 2 min, 4 min, 20 min and, for compound
2b only, 2 h after MDA addition (Fig. 3). To allow for this rapid
spectral acquisition following addition of MDA, neither
locking of the signal nor shimming was carried out. The reac-
tion of hydrazines 2a and 2b with MDA led to the rapid for-
mation (within 1 min) of new peaks associated with a putative
Scheme 2 Structure of MDA, colourimetric (6 → 7) and fluorogenic
probes (8a → 8b; 9a → 9b) used for its detection previously. The fluoro-
genic probe (2b → 4b) investigated in this work is structurally simpler
than previous probe examples.
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 3 Detailed 1H NMR analysis of the reactions between 5-MeO-N-aminoanthranilic acid (2a) and MDA (left), or methyl 5-MeO-N-aminoanthrani-
late (2b) and MDA (right). Spectra shown are before MDA addition (black), and <1 min (red), 2 min (green), 4 min (purple), 20 min (blue), and 2 h
(orange) after MDA addition. Brown and grey spectra are those of authentic standards 4a (left) or 4b (right).
pyrazole ring, as well as a slight change in benzene ring chemi- troscopy as read out. Fluorescence spectra associated with pyr-
cal shifts associated with starting materials 2a and 2b. Three azoles 4a and 4b were measured in 1× PBS, pH 7.2, containing
new signals at ca. 7.8 (HC), 7.7 (HA) and 6.5 ppm (HB) are 10% DMSO (Fig. ESI48 and 49†). Pyrazole 4a is a weak (Φ <
observed in both instances and their intensity increases with 0.02, harmane) blue fluorophore (λex 258 nm, λem 391 nm,
time (Fig. 3). The presence of the new signals is fully consist- Stoke’s shift 133 nm), which precluded a detailed investigation
ent with the formation of the pyrazole scaffold in both, 4a and of the reaction of 2a with MDA by fluorescence spectroscopy.
4b (see the Experimental and ESI† for the details), with only However, the replacement of the carboxylic acid moiety in pyr-
slight changes in chemical shifts (≤0.1 ppm) between the pro- azole 4b with the ester leads to a significant increase (>20 fold)
ducts of the reactions observed at the terminal time points (4a, in quantum yield (Φ 0.45, harmane)12 as well as a red shift of
20 min; 4b, 2 h) and corresponding pyrazole standards 4a and both excitation and emission wavelengths (4b, λex 307 nm, λem
4b having their spectra acquired under identical conditions.
410 nm, Stoke’s shift 103 nm), allowing for a detailed fluo-
This deviation in chemical shift is likely caused by the fact rescence-guided investigation of the reaction between methyl
that the spectrometer was not shimmed prior to the acqui- 5-MeO-N-aminoanthranilate (2b) and MDA.
sition of spectra during the time-course study. Consistent with
Upon mixing hydrazine 2b with MDA under illumination by
our previous observations,5b the reaction of hydrazine 2a with UV lamp (λex 365 nm), the evolution of blue-green fluorescence
MDA occurred very rapidly and only traces of the hydrazone was observed within 1 minute (Fig. 4a), which was red-shifted
intermediate can be observed as a small shoulder associated relative to the expected fluorescence of the synthesized pyra-
with the signal at ca. 7.7 ppm (HA) in the NMR spectra acquired zole standard, compound 4b (λem 410 nm). Detailed measure-
at earlier time points up until 4 min (Fig. 3, left). However, the ment of the fluorescence associated with the obtained mixture
signal observed at 7.7 ppm (HA) was of particular interest in the revealed two fluorescent species in solution: one with spectral
NMR spectra associated with hydrazine 2b wherein the signal is properties expected from compound 4b (λex 307 nm, λem
broad and unresolved even after 20 min of reaction, and a dis- 410 nm), and the other with λex 371 nm as an optimal exci-
tinct doublet is observed after 2 h (Fig. 3, right). This observation tation wavelength and associated emission at λem478 nm
prompted us to propose that a short-lived hydrazone intermedi- (Stoke’s shift 107 nm, Fig. 4b). When the mixture was left at
ate is present in solution and observable by NMR spectroscopy.
room temperature (rt) for 30 minutes, the initially observed blue-
In order to further investigate the formation and fate of the green fluorescence was no longer visible, but instead the mixture
proposed hydrazone intermediate observed by 1H NMR spec- produced a deep blue fluorescence with photophysical properties
troscopy, we performed an identical experiment to that per- matching those of 4b. Fluorescence kinetic measurements were
formed for the NMR study however using fluorescence spec- carried out that recapitulated these qualitative observations
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 4 Methyl 5-MeO-N-aminoanthranilate (2b) as a fluorogenic probe for the detection of MDA. (a) MDA was added to a 25 μM PBS solution of 2b
(pH 7.2) under illumination at 365 nm, and photographed at indicated time points. (b) Emission spectra of 1 μM 2b (black), or 2b after 1 min incu-
bation with 5 eq. MDA at 37 °C and excited at 310 nm (blue) or 375 nm (cyan). (c) Formation and rapid decay of short lived intermediate 10 formed in
the reaction between methyl 5-MeO-N-aminoanthranilate (2b) and MDA followed by fluorescence spectroscopy with λex/λem = 375/480 nm.
Kinetics of 25 μM 2b alone (squares) and 2b + MDA (circles) are shown, with three individual trials of 2b + MDA shown (light, medium, and dark
cyan).
(Fig. 4c). An initial formation of the blue-green fluorescent
species takes place rapidly, followed by a rapid decrease of the
fluorescent signal at λem 480 nm by 50% within 5 minutes, and
then a slower decrease over a longer period of time.
Considering the results of the fluorescence-based kinetic
study along with those from the detailed NMR experiment, a
mechanism for the reaction of 2b with MDA is proposed
(Scheme 3). While we believe that an identical mechanism
operates in the case of the transformation 2a → 4a supported
by the NMR study, we were unable to support this hypothesis
Fig. 5 The formation of the fluorescent hydrazone intermediate 10 is
dependent upon MDA concentration. (a) A 1 μM solution of 2b in PBS
by fluorescence measurements. The reaction of 2b with MDA
leads to the formation of the hydrazone intermediate 10,
which is transiently stabilized by the presence of a hydrogen
bond between the terminal hydrazone nitrogen and the
hydroxyl group of the enol tautomer of intermediate 10. The
enol tautomer would be favoured by the intramolecular hydro-
gen bond resulting in a pseudo-aromatic six-membered ring.
The nucleophilic attack of the ring-proximal nitrogen group
present in the hydrazone moiety onto the electrophilic carbo-
nyl would then take place, ultimately leading to the formation
of pyrazole 4b (Scheme 3). Importantly, the intensity of the
observed blue-green emission peak was dependent on MDA
concentration, supporting the hypothesis that the observed
fluorescence was associated with a semi-stable reaction inter-
mediate 10 (Fig. 5).
(pH 7.2) was mixed with the indicated molar equivalents of MDA and an
emission scan was recorded immediately after mixing using λex 375 nm
(blue curves). (b) Emission scans for MDA alone (open circles), 2b alone
(red), and the pyrazole standard 4b (black) are also shown. Note the
difference in the y-axis scaling between panels a and b.
Cyclization of methyl 5-MeO-N-aminoanthranilate (2b) in PBS
buffer
During our initial experiments involving solutions of methyl
5-MeO-N-aminoanthranilate dihydrochloride (2b) in PBS, we
noted the evolution of deep-blue fluorescence over 5 min from
an initially non-fluorescent probe stock solution. To under-
stand this phenomenon, we evaluated the stability of hydra-
zine 2b in PBS at 37 °C (Fig. 6). A significant (∼10 fold) fluo-
rescence signal increase (λem 421 nm) was observed within
Scheme 3 Reaction between hydrazine 2b and MDA proceeds via short lived intermediate 10.
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
of 2b can be significantly limited by preparation of stock solu-
tions in DMSO and guarding them from light, where cycliza-
tion to 11 was found to be slow (<5% conversion after 24 h) as
indicated by 1H NMR inspection.
Specificity of fluorogenic response of hydrazone 2b to biogenic
carbonyls, metabolites, and ions
In addition to MDA, we assayed the fluorogenic response of hydra-
zine 2b towards endogenous carbonyl compounds, including ali-
phatic aldehydes (formaldehyde, acetaldehyde, pentanal, hexanal,
octanal, nonanal), more complex aldehyde metabolites (glyoxal,
methylglyoxal, crotonaldehyde, glyceraldehyde), cofactors (pyridoxal
phosphate), ketones (acetone, pyruvate, butanedione, acetylace-
tone), glutathione, and biogenic cations (Ca2+, Cu2+, Fe3+, Mg2+,
Mn2+, Zn2+, Fig. 7). Among the carbonyls examined, hydrazine 2b
showed limited fluorescence signal turn-on in the presence of
ketones, glutathione and some complex aldehydes (crotonaldehyde,
glyoxal, pyridoxalphosphate). While some biogenic metal cations
(Ca2+, Fe3+, Mg2+) appeared to produce negligible fluorescence in
the presence of 2b, a moderate fluorescence signal turn-on is
observed from Cu2+, Mn2+ and Zn2+ (Fig. 7). This weak fluorescence
response is possibly attributable to the suitability of both the hydra-
zino- and methyloxycarbonyl groups as ligands for Cu2+, Mn2+ and
Zn2+, possibly promoting the formation of metal complexes in solu-
tion. In addition to glyceraldehyde and methylglyoxal, simple ali-
phatic aldehydes (formaldehyde, acetaldehyde, pentanal, hexanal,
octanal, and nonanal) showed a substantial fluorescence signal
turn-on upon reaction with hydrazine 2b (Fig. 7).
From a diagnostic standpoint, increased concentrations of
pentanal, hexanal, octanal and nonanal were found in the
exhaled breath of lung cancer patients when compared to
healthy smoker and non-smoker volunteers.8 While the for-
mation of hexanal results from the oxidative cleavage of lino-
leic or arachidonic acid26 and hexanal is considered a general
marker of oxidative stress,27 other aldehydes might also result
from lipid peroxidation if specific unsaturated fatty acids are
present in tumour cell membranes. Derivatization of these ali-
Fig. 6 Cyclization of methyl 5-MeO-N-aminoanthranilate (2b) in PBS
solution. Probe 2b was incubated at 37 °C in PBS (pH 7.2) for 1 (blue),
5 (red), and 20 min (green), and an emission scan was recorded with
λex 310 nm. The emission spectrum of the standard indazole 11 is shown
for comparison.
5 minutes of incubation, which continued to increase over
time. It is known that heating N-aminoanthranilic acids (e.g.
2a) in concentrated HCl results in the formation of inda-
zoles.25 This reaction involves a strong mineral acid-catalyzed
intramolecular nucleophilic attack of the terminal hydrazine
nitrogen onto the carbonyl group with the formation of a tetra-
hedral intermediate, followed by its collapse and subsequent
aromatization to afford blue fluorescent indazoles. Our
hypothesis (Fig. 6) was that a similar reaction was taking place
when hydrazine 2b is dissolved in PBS buffer. Although the
solution of 2b is neutral (pH ∼7.2), the concentration of 2b is
low enough (10−6 M) to favour intramolecular reactions.
Importantly, the ester moiety makes the carbonyl group in 2b
more prone to intramolecular nucleophilic attack by the term-
inal hydrazine nitrogen since methanol is a relatively better
leaving group, facilitating the collapse of the tetrahedral inter-
mediate. We have prepared indazole 11 (in 31% yield, Fig. 6)
by heating 5-MeO-N-aminoanthranilic acid (2a) according to a
previous method,25 and compared the fluorescence emission
spectra (λem 421 nm) of 11 to those obtained by simple incu-
bation of 2b in PBS. As shown in Fig. 6, a good match in emis-
sion maxima between standard 11 and stock solution contain-
ing 2b was observed upon excitation at 310 nm. Excitation at
higher wavelengths (λex 375 nm) required to induce hydrazone
fluorescence, resulted in background levels of fluorescence
emission from the indazole (Fig. ESI50a†). Furthermore, the
pseudo-first order rate constant for the cyclization reaction in
PBS (kobs = 0.009 0.002 min−1) was 10-fold lower than that for
hydrazone formation with hexanal as a test aldehyde (kobs
=
Fig. 7 Methyl 5-MeO-N-aminoanthranilate (2b) shows broad specificity
to aliphatic aldehydes. The fold fluorescence enhancement relative to
2b alone was recorded with λex/λem 375/480 nm following 1 min of incu-
bation of 1 μM solutions of 2b in PBS (pH 7.2) with 5 eq. of MDA (black
bar), simple aliphatic aldehydes (red bars), dialdehydes and more
complex aldehydes (blue bars), ketones (purple bars), glutathione
0.100 0.002 min−1) as determined by previously published
methods (Fig. ESI50b†).5 Together, this suggests that indazole
formation, while limiting levels of 2b in PBS, would not result
in artifactual fluorescence emission upon aldehyde sensing
with compound 2b. Importantly, the spontaneous cyclization (yellow bar) and biogenic metal cations (orange bars).
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Organic & Biomolecular Chemistry
Paper
phatic aldehydes as biomarkers of lung cancer and analysis by major source of aliphatic aldehydes is the decomposition of
fluorescent methods may present an alternative to tandem gas the cell membrane due to lipid peroxidation.14 Since the
chromatography mass spectrometry (GC-MS) analysis currently fluorogenic response of 2b to aldehydes spans a broad range
used.8,28 Compared to GC-MS analysis requiring expensive instru- of potential products of lipid peroxidation to yield fluorescent
mentation, as well as sample processing leading to potential inac- hydrazones with spectral properties amenable to conventional
curacies, fluorescence assays are significantly easier to perform fluorescence microscopy (i.e. λex 370 nm, λem 480 nm), we
and may be better suited to the point-of-care determination of sought to apply this probe to live cell imaging. A well-charac-
longer chain aliphatic aldehydes. Future studies will seek to apply terized model of lipid peroxidation was implemented, com-
fluorescence-based aldehyde fingerprinting that uses 2b as deriva- prising human HEK293 cells treated with diethyl maleate
tizing agent for the identification of hydrazones 3f–3i in complex (DEM), which perturbs cellular redox status with the endpoint
mixtures to rapidly determine their identities with a low-cost tech- of cell membrane catabolism through oxidative lipid degra-
nique.12 Overall our results indicate that hydrazine 2b could serve dation, and/or with α-tocopherol (α-TOH), nature’s most
as a fluorogenic probe for Aldehydic Load, providing for the effective antioxidant previously shown to limit DEM-induced
detection of a range of aliphatic aldehydes and MDA, which com- lipid peroxidation.29 Cells were either left untreated (Fig. 8b, g
prise catabolic products of lipid peroxidation.
and l), or were treated with 1.5 mM DEM for 1 h (Fig. 8c, h
and m) or 2 h (Fig. 8d, i and n) in order to induce lipid peroxi-
dation prior to incubation with 2b for 5 min. Micrographs
show limited fluorescence in untreated HEK293 cells,
which is expected due to a homeostatic flux of metabolic alde-
hydes and carbonyls such as glyceraldehyde and pyruvate.
However, a substantial fluorescence enhancement is observed
Imaging Aldehydic Load changes due to lipid peroxidation by
live cell microscopy
While there are a variety of biochemical processes that can
result in the production of aldehydes in cells and tissues, the
Fig. 8 Live cell microscopy of HEK293 cells incubated with diethyl maleate (DEM) to induce lipid peroxidation, and/or α-tocopherol (α-TOH) as an
anti-oxidant. HEK293 cells were either left untreated (b, g, l), treated with 1.5 mM diethyl maleate for 1 h (c, h, m) or 2 h (d, i, n), or treated with
α-tocopherol (10 mM, 24 h) (a, f, k) alone or prior to DEM (1.5 mM, 1 h) (e, j, o) and were then incubated with 100 μM 2b for 5 min prior to imaging.
The fluorescence micrographs overlayed on DIC images for the full field of view (top), as well as a zoom region (middle) are shown. Zoom region is
indicated by black dashed boxes. Heat map overlay of the fluorescence intensity shows relative distribution of imaging signal intensity in HEK293
cells (bottom). Scale bars for panels a–e = 100 μm, and panels f–o = 25 μm. All images were acquired with 405 nm excitation laser, and an emission
window of 420 to 520 nm.
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
after 1 h of DEM treatment in some cells, which continues to uniqueness of methyl 5-MeO-N-aminoanthranilate derives from
increase in both intensity and frequency after 2 h of incu- its minimal nature: facile synthesis for easy accessibility, simple
bation. In order to reduce aldehydic load, HEK293 cells were wash-free implementation for live cell microscopy, and broad
treated with 10 μM α-TOH for 24 h and left untreated (Fig. 8a, f aliphatic aldehyde binding for total aldehyde load mapping. In
and k) or treated with 1.5 mM DEM for 1 h (Fig. 8e, j and o) effect, the minimalism of the probe maximizes its application
prior to incubation with 2b for 5 min. A reduction of aldehydic to evaluate the chemical biology of carbonyls. Future studies
load was observed following both treatments, with α-TOH will involve chemical modifications of 2b to prevent the
reducing DEM-induced aldehyde production, as well as spontaneous cyclization in PBS, the formation of fluorescent
homeostatic aldehyde production in the cultured cells. molecules emitting at longer wavelengths (λem > 500 nm), and
Although the specific identity of the aldehydes contributing to the application of 2b to interrogate the chemical biology of
the fluorescence enhancement observed in HEK293 cells endogenous aldehydes in cellular models of disease.
cannot be determined using 2b, the broad reactivity of the
probe for aliphatic aldehydes in general provides a good
measure of Aldehydic Load, an important emerging biomarker
Experimental
General experimental procedures
for studying a range of diseases.30
Reagents were commercially available, except for 5-MeO-N-ami-
noanthranilic acid (2a),5b methyl 5-MeO-N-aminoanthranilate
Conclusions
dihydrochloride (2b),12 pyrazoles 4a, 4b12 and indazole 1125
which were prepared according to the literature procedures. All
solvents were HPLC grade except for water (18.2 MΩ cm milli-
pore water). Solvents were removed under reduced pressure in
a rotary evaporator, aqueous solutions were lyophilized and
organic extracts were dried over Na2SO4. Flash column chrom-
atography (FCC) was carried out using silica gel (SiO2), mesh
size 230–400 Å. Thin-layer chromatography (TLC) was carried
out on Al backed silica gel plates with compounds visualised by
anisaldehyde stain, 5% ninhydrin stain, and UV light. NMR
spectra were recorded on a 300 or 400 MHz spectrometers, for
1H NMR spectra δ values were recorded as follows: DMSO-D6
(2.50 ppm); for 13C (93.75 or 125 MHz) δ DMSO-D6 (39.50 ppm).
Mass spectra (MS) were obtained using electron impact (EI).
Steady state fluorescence spectra were acquired on a Cary
Eclipse fluorescence spectrophotometer, using 1× PBS, pH 7.2
containing up to 10% DMSO. Quantum yields were determined
as described previously,31 using harmane as a standard.13
The use of methyl 5-MeO-N-aminoanthranilate dihydrochlor-
ide (2b) as a fluorogenic probe capable of detecting endogen-
ous aldehydes was examined in detail. The current work comp-
lements a previous description of 2b in the context of fluo-
rescent fingerprinting of aldehydes,12 and seeks to fully
describe its intra- and intermolecular reactivity, as well as the
utility of 2b for live cell microscopy of aldehydic load. The reac-
tion of 2b with MDA (formed in situ from MDA tetramethyl
acetal) was found to produce a blue fluorescent pyrazole 4b
(λem 410 nm, Φ 0.45), proceeding through a blue-green fluo-
rescent hydrazone intermediate 10. The simple structures of
both 2b and 4b facilitated a detailed 1H NMR-based mechanis-
tic evaluation of the reaction, presenting evidence of the hydra-
zone intermediate that was also supported by fluorescence
spectroscopy studies. The selectivity of 2b toward the detection
of cellular components was also evaluated, revealing that in
addition to MDA,
a significant fluorogenic response is
observed with simple aliphatic aldehydes. Longer chain ali-
phatic aldehydes, produced, for example, during lung cancer
progression, led to the formation of fluorescent hydrazones
Reaction of 2b with aliphatic aldehydes (pentanal, hexanal,
octanal, nonanal)
(3f–3i), emitting in the “far” blue region (λem 466–480 nm). Aliphatic aldehydes (pentanal, 53 μL; hexanal, 61 μL; octanal,
Quantum yields associated with these fluorophores were 78 μL; nonanal, 86 μL; 0.5 mmol each) were added to separated
found to be modest (Φ 0.06–0.17), attributable to the occur- stirred solutions of methyl 5-MeO-N-aminoanthranilate di-
rence of hydrazones as inseparable mixtures of E- and hydrochloride (2b, 132 mg, 0.5 mmol) in MeOH (5 mL) and
Z-isomers. One caveat for the use of 2b for Aldehydic Load water (1.25 mL). The mixtures were stirred for 18 h at rt;
imaging derives from the instability of hydrazine 2b in MeOH was evaporated, the residues were diluted with brine
aqueous solution and the minute time-scale lifetime of the (30 mL) and were extracted with EtOAc (2 × 30 mL). Combined
hydrazone intermediate 10, limiting detection reliability to organic extracts were dried, were concentrated and the resi-
within 5 min of probe application. Even with this caveat in dues were subjected to FCC on 40 g SiO2 as follows: hydrazone
place, Aldehydic Load enhancement following oxidative stress- 3f, hexanes/EtOAc (4 : 1); hydrazones 3g–3i, hexanes/EtOAc
induced lipid peroxidation was successfully imaged through (9 : 1). Evaporation of the eluates afforded hydrazones 3f–3i.
live cell microscopy of HEK293 cells treated with DEM. Overall
Hydrazone 3f. Yellow oil, mixture of E- and Z-isomers,
our results present a detailed evaluation of the reaction 110 mg, 83%. 1H NMR δ (DMSO-D6) 10.51 (s, D2O exch., 0.4H);
between a fluorogenic hydrazine-derived probe and MDA, and 10.33 (s, D2O exch., 0.6H); 7.53 (d, J = 9.0 Hz, 0.4H); 7.43 (d, J =
might provide a useful guideline for the development of rapid 9.0 Hz, 0.6H); 7.37 (t, J = 5.0 Hz, 0.6H); 7.32 (d, J = 3.0 Hz,
and easy to perform fluorescence assays for point-of-care detec- 0.4H); 7.27 (d, J = 3.0 Hz, 0.6H); 7.18 (dd, J = 9.0, 3.0 Hz, 0.4H);
tion of Aldehydic Load relevant to a variety of diseases. The 7.14 (dd, J = 9.0, 3.0 Hz, 0.6H); 6.59 (t, J = 5.0 Hz, 0.4H); 3.86
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Organic & Biomolecular Chemistry
Paper
(s, 1.2H); 3.83 (s, 1.8H); 3.72 (s, 1.2H); 3.71 (s, 1.8H); 2.23 (m, 1H NMR spectra were acquired. The tubes were then removed
2H); 1.45 (m, 4H); 0.90 (m, 3H). 13C NMR δ (DMSO-D6) 167.9, from the spectrometer, 1 drop of 1 M solution of MDA tetra-
167.4, 150.9, 150.4, 144.2, 142.8, 142.4, 142.3, 123.2 (2 × C), methyl acetal in 1 M HCl (known to form MDA in situ)24 was
114.8, 114.7, 112.6 (2 × C), 108.8, 108.1, 55.4 (2 × C), 52.2, 51.9, added and 1H NMR spectra were acquired immediately (1 min)
31.5, 28.5, 27.5, 26.1, 22.0, 21.7, 13.8, 13.7. HRMS (EI) m/z; and then after 2, 4, 20 and 120 (2b only) minutes. Separate
found 264.1473 [M+] (calcd 264.1474 for C14H20N2O3), LRMS solutions of pyrazoles 4a, 4b and MDA (controls) were pre-
(EI) m/z (rel. abundance): 264 [M+] (100), 180 (87), 164 (24), pared and analyzed under identical conditions.
150 (49), 122 (53).
Fluorescence study. A 10 mM stock solution of 2b was pre-
Hydrazone 3g. Yellow oil, mixture of E- and Z-isomers, pared in DMSO, and 5 µL was added to 1× PBS, pH 7.2, in a
1
85 mg, 61%. H NMR δ (DMSO-D6) 10.51 (s, D2O exch., 0.4H); 1 cm path length quartz cuvettes, followed by mixing by inver-
10.33 (s, D2O exch., 0.6H); 7.53 (d, J = 9.0 Hz, 0.4H); 7.47 (d, J = sion. To this solution, 50 μL of 1 M solution of MDA tetra-
9.0 Hz, 0.6H); 7.37 (t, J = 5.0 Hz, 0.6H); 7.32 (d, J = 3.0 Hz, methyl acetal in 1 M HCl was added,24 immediately followed
0.4H); 7.27 (d, J = 3.0 Hz, 0.6H); 7.18 (dd, J = 9.0, 3.0 Hz, 0.4H); by 5 μL of 10 M NaOH to neutralize the solution (as confirmed
7.14 (dd, J = 9.0, 3.0 Hz, 0.6H); 6.58 (t, J = 5.0 Hz, 0.4H); 3.86 by a pH paper). Fluorescence emission was recorded on a fluo-
(s, 1.2H); 3.83 (s, 1.8H); 3.72 (s, 1.2H); 3.71 (s, 1.8H); 2.22 (m, rescence spectrophotometer with λex/λem 310/410 nm and
2H); 1.53 (m, 2H); 1.33 (m, 4H); 0.89 (m, 3H). 13C NMR 375/475 nm and slit widths of 5 nm. Readings were acquired
δ (DMSO-D6) 167.9, 167.4, 150.9, 150.4, 144.2, 142.8, 142.4, concurrently every 10 s for 45 min, the experiment was
142.3, 123.2, 123.1, 114.8, 114.7, 112.7, 112.6, 108.8, 108.2, repeated three separate times.
55.4 (2 × C), 52.2, 51.9, 31.7, 31.0, 30.8, 26.3, 26.0, 25.0, 21.9,
21.8, 13.9, 13.8. HRMS (EI) m/z; found 278.1638 [M+] (calcd
Hydrazine 2b as a fluorogenic probe for Aldehydic Load
278.1630 for C15H22N2O3), LRMS (EI) m/z (rel. abundance): 278
[M+] (100), 180 (83), 164 (43), 150 (37), 122 (38).
Detection of MDA. The samples were prepared as follows:
100 μL of the solution of 2b (10−5 M) in DMSO was mixed with
Hydrazone 3h. Yellow oil, mixture of E- and Z-isomers, 1× PBS (pH 7.2) and a stock solution of MDA24 was added to
119 mg, 78%. 1H NMR δ (DMSO-D6) 10.51 (s, D2O exch., 0.4H); achieve the final volume of 1 mL (final concentration of 2b in
10.32 (s, D2O exch., 0.6H); 7.53 (d, J = 9.0 Hz, 0.4H); 7.47 (d, J = the sample is 10−6 M). Seven separate samples were prepared,
9.0 Hz, 0.6H); 7.37 (t, J = 5.0 Hz, 0.6H); 7.32 (d, J = 3.0 Hz, the concentrations of MDA were 10−7 M (0.1 eq.), 2 × 10−7
M
0.4H); 7.28 (d, J = 3.0 Hz, 0.6H); 7.18 (dd, J = 9.0, 3.0 Hz, 0.4H); (0.2 eq.), 5 × 10−7 M (0.5 eq.), 10−6 M (1 eq.), 2 × 10−6 M (2 eq.),
7.14 (dd, J = 9.0, 3.0 Hz, 0.6H); 6.59 (t, J = 5.0 Hz, 0.4H); 3.86 5 × 10−6 M (5 eq.), 8.5 × 10−6 M (8.5 eq.). The pH in the
(s, 1.2H); 3.83 (s, 1.8H); 3.72 (s, 1.2H); 3.71 (s, 1.8H); 2.23 (m, samples containing larger amounts of MDA stock solution
2H); 1.53 (m, 2H); 1.32 (m, 8H); 0.85 (m, 3H). 13C NMR (>2 eq.) was adjusted (to reach ∼pH 7.2) by the addition of satu-
δ (DMSO-D6) 167.9, 167.4, 150.9, 150.4, 144.2, 142.8, 142.4, rated NaHCO3 solution (10–20 μL). Control samples containing
142.3, 123.2, 123.1, 114.8, 114.6, 112.6 (2 × C), 108.8, 108.1, just hydrazine 2b, pyrazole 4b and MDA (final concentration
55.4 (2 × C), 52.1, 51.9, 31.8, 31.2, 31.1, 28.8, 28.6, 28.5, 28.4, of 10−6 M) were also prepared. Each sample was analyzed right
26.4, 26.3, 25.4, 22.1 (2 × C), 13.9 (2 × C). HRMS (EI) m/z; after preparation (within
1 min) by fluorescence scans
found 306.1953 [M+] (calcd 306.1943 for C17H26N2O3), LRMS (λex 371 nm or 307 nm). All measurements were performed at
(EI) m/z (rel. abundance): 306 [M+] (100), 180 (79), 164 (34), 37 °C. The same temperature was maintained to keep the MDA
150 (36), 122 (40).
stock solution as well as PBS. The stock solution of hydrazine
Hydrazone 3i. Yellow oil, mixture of E- and Z-isomers, 2b in DMSO was stored at rt in the dark (flask wrapped in Al
119 mg, 74%. 1H NMR δ (DMSO-D6) 10.51 (s, D2O exch., 0.4H); foil). Bell-type curves were fitted to raw data points of emission
10.32 (s, D2O exch., 0.6H); 7.52 (d, J = 9.0 Hz, 0.6H); 7.47 (d, J = scans using standard protocols in GraphPad Prism v.7.0c.
9.0 Hz, 0.4H); 7.37 (t, J = 5.5 Hz, 0.4H); 7.32 (d, J = 3.0 Hz,
Response to other carbonyls, metal ions and biologically
0.6H); 7.27 (d, J = 3.0 Hz, 0.6H); 7.18 (dd, J = 9.0, 3.0 Hz, 0.6H); relevant molecules. The samples containing 2b (final concen-
7.14 (dd, J = 9.0, 3.0 Hz, 0.4H); 6.59 (t, J = 5.5 Hz, 0.6H); 3.86 tration 10−6 M) were prepared as described above. A solution
(s, 1.8H); 3.83 (s, 1.2H); 3.72 (s, 1.8H); 3.70 (s, 1.2H); 2.22 (m, of 5 eq. of the analyte of interest was added (final concen-
2H); 1.53 (m, 2H); 1.36 (m, 10H); 0.85 (m, 3H). 13C NMR tration 5 × 10−6 M), the fluorescence scans (λex 371 nm) were
δ (DMSO-D6) 167.9, 167.4, 151.0, 150.5, 144.2, 142.8, 142.4 (2 × performed as described above.
C), 123.2, 123.1, 114.8, 114.7, 112.7, 112.6, 108.8, 108.2, 55.4
Instability of 2b in PBS. A solution of hydrazine 2b in DMSO
(2 × C), 52.1, 51.9, 31.8, 31.3, 31.2, 28.9, 28.8, 28.7, 28.6 (3 × C), (100 μL) was mixed with PBS (overall volume 1 mL, final con-
26.4, 26.3, 25.4, 22.1 (2 × C), 13.9 (2 × C). HRMS (EI) m/z; centration 10−6 M, 37 °C). The fluorescence scans (λex 327 nm)
found 320.2081 [M+] (calcd 320.2100 for C18H28N2O3), LRMS were performed immediately (1 min), then after 5 and 20 min.
(EI) m/z (rel. abundance): 320 [M+] (32), 180 (52), 164 (19), 150 A sample containing indazole 11 was prepared and analyzed
(22), 122 (21).
under identical conditions.
Live-cell microscopy study. HEK293 cells were a gift from the
lab of Dr D. A. Pratt (University of Ottawa). Cells were cultured
Reaction of 2a and 2b with MDA – detailed kinetic analysis
NMR study. Separate solutions of 2a or 2b (1 mg each) in 1× in Minimal Essential Medium supplemented with 10% v/v
PBS, pH 7.2 containing 10% DMSO-D6 were prepared and the Fetal Calf Serum, 1% v/v Pen Strep antibiotic solution, and 1%
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
MEM non-essential amino acid solution at 37 °C in a 5% CO2
atmosphere. Cells were plated at 25 000 cells per well in a
24-well tissue culture-treated polystyrene plate overnight prior to
use for microscopy. Growth medium was changed prior to treat-
ment with 10 μM α-TOH for 24 h, or 1.5 mM DEM for 1 and 2 h.
At indicated time points, media was removed and replaced with
1× Hank’s Balanced Salt Solution, pH 7.2, and 10 μL of a solution
of 2b in DMSO (final concentration of 2b = 100 μM) was added.
Imaging was performed on a Zeiss LSM 880 confocal microscope
fitted with live cell incubator chamber using 405 nm excitation
laser line, and 10x objective lens. Both, differential interference
contrast (DIC) and fluorescence images were acquired. All image
analysis was performed using ImageJ v1.45S.
5 (a) E. T. Kool, D. H. Park and P. Crisalli, J. Am. Chem. Soc.,
2013, 135, 17663; (b) T. Dang, M. Suchý, Y. J. Truong,
W. Oakden, W. W. Lam, C. Lazurko, G. Facey, G. J. Stanisz
and A. J. Shuhendler, Chem. – Eur. J., 2018, 24, 9148.
6 P. J. O’Brien, A. G. Siraki and N. Shangari, Crit. Rev.
Toxicol., 2005, 35, 609.
7 O. E. Owen, V. E. Trapp, C. L. Skutches, M. A. Mozzoli,
R. D. Hoeldtke, G. Boden and G. A. Reichard Jr., Diabetes,
1982, 31, 242.
8 P. Fuchs, C. Loeseken, J. K. Schubert and W. Miekisch,
Int. J. Cancer, 2010, 126, 2663.
9 D. T. Antoniak, M. J. Duryee, T. R. Mikuls, G. M. Thiele and
D. R. Anderson, Free Radicals Biol. Med., 2015, 89, 409.
10 S. Ivanova, G. I. Botchkina, Y. Al-Abed, M. Meistrell III,
F. Batliwalla, J. M. Dubinsky, C. Iadecola, H. Wang,
P. K. Gregersen, J. W. Eaton and K. J. Tracey, J. Exp. Med.,
1998, 188, 327.
Conflicts of interest
The authors declare no competing financial interest.
11 For selected examples see; (a) T. F. Brewer and C. J. Chang,
J. Am. Chem. Soc., 2015, 137, 10886; (b) L. H. Yuen,
N. S. Saxena, H. S. Park, K. Weinberg and E. T. Kool, ACS
Chem. Biol., 2016, 11, 2312; (c) T. F. Brewer, G. Burgos-
Barragan, N. Wit, K. J. Patel and C. J. Chang, Chem. Sci., 2017,
8, 4073; (d) K. J. Bruemmer, O. Green, T. A. Su, D. Shabat and
C. J. Chang, Angew. Chem., Int. Ed., 2018, 57, 7508; (e) M. Yang,
J. Fan, J. Zhang, J. Du and X. Peng, Chem. Sci., 2018, 9, 6758.
12 C. Lazurko, I. Radonjic, M. Suchý, G. Liu, A. G. Rolland-
Lagan and A. J. Shuhendler, ChemBioChem, 2018, DOI:
10.1002/cbic.201800427, in press.
Acknowledgements
We wish to acknowledge Professor Jeffrey Keillor for allowing us
to use his Cary Eclipse Fluorescence Spectrophotometer for the
duration of this work. This work was supported by an NSERC
Discovery Grant RGPIN 2015-05796 (A. J. S.), the Canada
Research Chairs Program 950-230754 (A. J. S.), and the Canadian
Institutes of Health Research PJT376892 (A. J. S.); the financial
support provided by these agencies is gratefully acknowledged.
13 A. Pardo, D. Reyman, J. M. L. Poyato and F. Medina,
J. Lumin., 1992, 51, 269.
14 Z. A. M. Zielinski and D. A. Pratt, J. Org. Chem., 2017, 82, 2817.
15 MDA is known to form adducts with DNA rapidly, see for
example in. (a) D. Pluskota-Karwatka, A. J. Pawlowicz,
M. Bruszyńska, A. Greszkiewicz, R. Latajka and
L. Kronberg, Chem. Biodiversity, 2010, 7, 959; (b) K. Salus,
M. Hoffmann, T. Siodla, B. Wyrzykiewicz and D. Pluskota-
Karwatka, New J. Chem., 2017, 41, 2409.
Notes and references
1 O. K. Abou-Zied, B. Y. Al-Busaidi and J. Husband, J. Phys.
Chem. A, 2014, 118, 103.
2 According to a recent review, fluorescence properties
associated with anthranilates have never been integrated
into imaging probe development see: (a) R. Duval and
C. Duplais, Nat. Prod. Rep., 2017, 34, 161. For an isolated 16 A. Gönenç, Y. Ozkan, M. Torun and B. Simşek, J. Clin.
example exploiting the blue fluorescence of anthranilic Pharm. Ther., 2001, 26, 141.
acid within the context of chemical biology see: 17 M. Viigimaa, J. Abina, G. Zemtsovskaya, A. Tikhaze,
(b) C. Coburn, E. Allman, P. Mahanti, A. Benedetto,
F. Cabreiro, Z. Pincus, F. Mathijssens, C. Araiz, A. Mandel,
G. Konovalova, E. Kumskova and V. Lankin, Blood Press.,
2010, 19, 164.
M. Vlachos, S. A. Edwards, G. Fischer, A. Davidson, 18 L. S. Bir, S. Demir, S. Rota and M. Köseoğlu, Tohoku J. Exp.
R. E. Pryor, A. Stevens, F. J. Sack, N. Tavernarakis, Med., 2006, 208, 33.
B. P. Braeckman, F. C. Schroeder, K. Nehrke and D. Gems, 19 I. Hajimohammadreza and M. Brammer, Neurosci. Lett.,
PLoS Biol., 2013, 11, e1001613. 1990, 112, 333.
3 While the functionalization of anthranilic acids to form 20 V. Nair and G. A. Turner, Lipids, 1984, 19, 804.
anthranilamides is well developed, see.(a) N. Kanişkan, 21 D. R. Janero, Free Radicals Biol. Med., 1990, 9, 515.
Ş. Kökten and Ĭ. Çelik, ARKIVOC, 2012, viii, 198 and refer- 22 J. Chen, L. Zeng, T. Xia, S. Li, T. Yan, S. Wu, G. Qiu and
ences cited therein, the modification of the benzene ring to
allow for the conjugation of anthranilic acids by e.g. “click” 23 L. He, X. Yang, K. Xu and W. Lin, Chem. Commun., 2017,
reaction is underdeveloped. For an isolated example see: 53, 4080.
(b) C. S. McKay and M. G. Finn, Angew. Chem., Int. Ed., 24 Y. Tsuruta, Y. Date, H. Tonogaito, N. Sugihara, K. Furuno
2016, 55, 12643. and K. Kohashi, Analyst, 1994, 119, 1047.
4 (a) P. Crisalli and E. T. Kool, Org. Lett., 2013, 15, 1646; 25 L. Baiocchi, G. Corsi and G. Palazzo, Synthesis, 1978, 633.
Z. Liu, Anal. Chem., 2015, 87, 8052.
(b) P. Crisalli and E. T. Kool, J. Org. Chem., 2013, 78, 1184.
26 F. Shahidi, Adv. Exp. Med. Biol., 2001, 488, 113.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2018

View Article Online
Organic & Biomolecular Chemistry
Paper
27 M. Corradi, P. Pignatti, P. Manini, R. Andreoli, M. Goldoni, 29 B. Li, F. Zheng, J. R. Chauvin and D. A. Pratt, Chem. Sci.,
M. Poppa, G. Moscato, B. Balbi and A. Mutti, Eur. Respir. J.,
2004, 24, 1011.
28 D. Poli, M. Goldoni, M. Corradi, O. Acampa, P. Carbognani,
E. Internullo, A. Casalini and A. Mutti, J. Chromatogr. B:
Anal. Technol. Biomed. Life Sci., 2010, 878, 2643.
2015, 6, 6165.
30 (a) L. H. Yuen, N. S. Saxena, H. S. Park, K. Weinberg and
E. T. Kool, ACS Chem. Biol., 2016, 11, 2312; (b) E. M. Ellis,
Pharmacol. Ther., 2007, 115, 13.
31 D. F. Eaton, J. Photochem. Photobiol., B, 1988, 2, 523.
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem.