Deeper insight into protease-sensitive "covalent-assembly" fluorescent probes for practical biosensing applications

Article abstract of DOI:10.1039/c9ob01773a

We report a rational and systematic study devoted to the structural optimisation of a novel class of protease-sensitive fluorescent probes that we recently reported (S. Debieu and A. Romieu, Org. Biomol. Chem., 2017, 15, 2575-2584), based on the "covalent-assembly" strategy and using the targeted enzyme penicillin G acylase as a model protease to build a fluorescent pyronin dye by triggering a biocompatible domino cyclisation-aromatisation reaction. The aim is to identify ad hoc probe candidate(s) that might combine fast/reliable fluorogenic "turn-on" response, full stability in complex biological media and ability to release a second molecule of interest (drug or second fluorescent reporter), for applications in disease diagnosis and therapy. We base our strategy on screening a set of active methylene compounds (C-nucleophiles) to convert the parent probe to various pyronin caged precursors bearing Michael acceptor moieties of differing reactivities. In vitro stability and fluorescent enzymatic assays combined with HPLC-fluorescence analyses provide data useful for defining the most appropriate structural features for these fluorogenic scaffolds depending on the specifications inherent to biological application (from biosensing to theranostics) for which they will be used.

Full text of DOI:10.1039/c9ob01773a

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Deeper insight into protease-sensitive “covalent-
assembly” fluorescent probes for practical
biosensing applications†‡
Cite this: Org. Biomol. Chem., 2019,
17, 8918
Kévin Renault, a Sylvain Debieu,§a Jean-Alexandre Richard b and
a
Anthony Romieu
*
We report a rational and systematic study devoted to the structural optimisation of a novel class of pro-
tease-sensitive fluorescent probes that we recently reported (S. Debieu and A. Romieu, Org. Biomol.
Chem., 2017, 15, 2575–2584), based on the “covalent-assembly” strategy and using the targeted enzyme
penicillin G acylase as a model protease to build a fluorescent pyronin dye by triggering a biocompatible
domino cyclisation–aromatisation reaction. The aim is to identify ad hoc probe candidate(s) that might
combine fast/reliable fluorogenic “turn-on” response, full stability in complex biological media and ability
to release a second molecule of interest (drug or second fluorescent reporter), for applications in disease
diagnosis and therapy. We base our strategy on screening a set of active methylene compounds
(C-nucleophiles) to convert the parent probe to various pyronin caged precursors bearing Michael
acceptor moieties of differing reactivities. In vitro stability and fluorescent enzymatic assays combined
with HPLC-fluorescence analyses provide data useful for defining the most appropriate structural features
for these fluorogenic scaffolds depending on the specifications inherent to biological application (from
biosensing to theranostics) for which they will be used.
Received 12th August 2019,
Accepted 19th September 2019
DOI: 10.1039/c9ob01773a
rsc.li/obc
response (intensometric or ratiometric detection mode)
arising from selective interaction/reaction of the probe with its
Introduction
In the growing field of small molecule activatable (or “smart”) supposed target (bio)analyte.3 The ultimate goal is to improve
fluorescent probes for biological and environmental analysis detection sensitivity regardless of the complexity of the bio-
and bioimaging (i.e., biomedical applications related to in vivo logical/environmental matrices to be analysed.
molecular imaging, image-guided drug delivery and thera-
By the mid 2000s, a new probe design principle namely
nostics), innovation is a primary concern as evidenced by the “covalent-assembly” approach had emerged as a valuable
numerous valuable research studies published annually.1 It alternative to conventional pro-fluorophores based on the pro-
focuses mainly on both the development of high performance tection–deprotection of an optically tunable amino or hydroxyl
organic-based fluorophores2 and discovery of novel and group.4 Originally proposed by Anslyn and Yang,5 the basic
effective approaches/mechanisms to optimize the fluorogenic rationale of the “covalent-assembly” type probes is the for-
mation of a fluorophore via a covalent cascade reaction of two
fragments that most commonly proceeds in an intramolecular
aICMUB, UMR 6302, CNRS, Univ. Bourgogne Franche-Comté, 9,
Avenue Alain Savary, 21000 Dijon, France. E-mail: anthony.romieu@u-bourgogne.fr;
http://www.icmub.com
bFunctional Molecules and Polymers, Institute of Chemical and Engineering Sciences
(ICES), Agency for Science, Technology and Research (A*STAR), 8 Biomedical Grove,
Neuros, #07–01, Singapore 138665. E-mail: jean_alexandre@ices.a-star.edu.sg
†A preprint was previously posted on ChemRxiv, see https://doi.org/10.26434/
chemrxiv.9445202.v1
manner and is triggered by the species to be detected. The fun-
damental feature of the “covalent-assembly” probe is that it
guarantees both a colorimetric change (except for UV-absorb-
ing fluorophores such as traditional 7-N,N-dialkylamino or
7-hydroxycoumarins) and an optimal “turn-on” fluorescent
signal from a zero background.6 Alternatively, the “covalent-
assembly” process occurring from an “already-on” fluorescent
caged precursor may sometimes lead to a new electronic push–
pull conjugated backbone and thus causes a significant “red-
shift” of the fluorescence spectra particularly well-suited for
devising ratiometric detection schemes.7 The vast majority of
“covalent-assembly” type probes already published, more than
‡Electronic supplementary information (ESI) available: Experimental details
related to photophysical characterisation, fluorescence-based in vitro assays and
HPLC-fluorescence/-MS analyses and all analytical data of the synthesised com-
pounds. See DOI: 10.1039/c9ob01773a
§Present address: PSL Université Paris, Institut Curie, CNRS UMR 3666, INSERM
U1143, 75005 Paris France.
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60 examples dealing with the detection of a wide range of ana- type probes whose activation leads to the internal construc-
lytes including biothiols, enzymes, metal cations and ROS/ tion of xanthene dyes emitting in the range 550–625 nm. This
RNS, involve in situ formation of blue-green emitting 7-N,N- is a major achievement supported by in vitro fluorogenic detec-
dialkylamino/7-hydroxy-(2-imino)coumarins or related fluoro- tion assays of relevant analytes including: Sarin mimics and
phores through analyte-triggered lactonisation or Pinner cycli- Hg(II) cations (in situ formation of pyronin B),9 proteases
sation reactions.5a,8 To expand this innovative molecular namely penicillin G acylase (PGA) and leucine amino pepti-
sensing approach to longer-wavelength fluorophores, we along dase (LAP) (in situ formation of unsymmetrical pyronin AR116,
with the Yang group have recently designed “covalent-assembly” Fig. 1)10 and nitrogen dioxide NO2• (in situ formation of a rosa-
Fig. 1 State-of-the-art (structures and detection mechanism) “covalent-assembly” type probes for protease sensing through in situ formation of
unsymmetrical pyronin AR116. In magenta colour, the approach chosen in the present work for the rational optimisation of these probes, to find the
best compromise between reactivity, stability and versatility with the possible concomitant release of a second molecule of interest (Bn = benzyl,
EWG = electron-withdrawing group, PB = phosphate buffer).
Scheme 1 Synthesis of Michael acceptor-based PGA-sensitive probes 4–10 through the Knoevenagel condensation reaction. aFor the structure of
probe 7, see Fig. 5. Please note: probes 7–9 were isolated as TFA salts (EWG = electron-withdrawing group, FC (SiO2) = flash-column chromato-
graphy over silica gel, RT = room temperature). Please note: molecule numbering 4–10 corresponds to probe’s description and not C-nucleophiles
used for their preparation.
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mine).11 These encouraging results have convinced us that the rate of in situ pyronin formation was achieved by converting
“covalent-assembly” approach may have great potential to the formyl group of 2 into the dicyanomethylidenyl moiety
facilitate (1) the design of “smart” in vivo imaging agents (acti- (less than 1 h to achieve quantitative formation of AR116).14
vated by disease-associated enzymes)12 and (2) their possible However, the resulting PGA-sensitive probe 3 acts as a fluoro-
conversion to fluorogenic reaction-based prodrug conjugates gen with aggregation-induced emission (AIEgen)15 displaying
(i.e., theranostic agents)13 for diagnostic and therapeutic pur- an intense red fluorescence (emission centered at ca. 600 nm
poses. However, to achieve these ambitious goals, first of all it with a quantum yield of 6%) that prevents detection accord-
is essential to demonstrate that enzyme-triggered xanthene for- ing to an intensometric approach. Furthermore, its aqueous
mation is a versatile process, not negatively impacted by inter- stability and possible undesired reactivity with molecules cur-
ferences found in biological media (e.g., biothiols). Equally rently found in biological media have not yet been carefully
important will be the demonstration that kinetics can be easily studied. To address these items and to identify the best pro-
fine-tuned and the cascade mechanism can be readily applied tease-triggered reaction-based probe candidates that might
to the concomitant release of bioactive compounds. This will combine fast/reliable fluorogenic “turn-on” response, overall
entail significant efforts geared toward the structural optimiz- stability and ability to release a second molecule of interest
ation of caged precursors based on a mixed bis-aryl ether core (in addition to pyronin AR116), we have screened a short
structure10 (Fig. 1). To rapidly reach a representative range of library of C-nucleophiles whose reaction with aldehyde 2
such “covalent-assembly” type probes, the conversion of the should lead to a set of probes exhibiting their own intrinsic
formyl group (found in probes 1 and 2) to various Michael reactivity closely tied to the pKa value of the nucleophilic
acceptor moieties through Knoevenagel condensation may well partner involved in their synthesis. To cover a broad range of
be the preferred route (Scheme 1). Indeed, numerous latent C-nucleophiles that fulfills the requirements mentioned
C-nucleophiles are stable, cheap and commercially available above, we have chosen to use both commercial basic organic
and their structural diversity (related to their pKa value) offers building blocks and less conventional molecular structures:
a unique opportunity for adjusting the subtle balance between dimedone (pKa 5.2 in water),16 1,3-dimethylbarbituric acid
the stability and reactivity of the probes under aqueous physio- (pKa 4.7 water),17 Meldrum’s acid (pKa 4.8 in water),16,18
logical conditions. Furthermore, such reactive moieties may be 4,7-dihydroxycoumarin (pKa 4.4 in water, see the ESI‡ for its
found in molecules either displaying a specific biological photophysical characterization in phosphate buffer (PB)),19
activity (e.g., benzodiazepinone and 5-pyrazolone derivatives) 1,4-dimethylpyridinium iodide (pKa never determined), 1-ethyl-
and/or acting as a second fluorescent reporter (e.g., 4,7- 2,3,3-trimethylindolenium iodide (pKa never determined),
dihydroxycoumarin).
edaravone (a free radical scavenger and neuroprotective agent
Herein, we report the synthesis of eight different PGA-sensi- used for the therapy of amyotrophic lateral sclerosis, also
tive “covalent-assembly” fluorogenic probes whose Michael known as 1-phenyl-3-methyl-5-pyrazolone, pKa 7.0 in water)20
acceptor moiety stems from parent C-nucleophiles covering a and NSC 645039 (4-phenyl-1,3-dihydro-2H-1,5-benzodiazepin-2-
broad range of pKa values (from 4.4 to 12.7). Their fluorogenic one, pKa 12.7, predicted value),21 compared with malononitrile
behavior as well as their enzymatic activation and aqueous (pKa 11.5).22
stability were studied in detail through in vitro fluorescence
assays and HPLC-fluorescence/-MS analyses. All data generated
have been used to establish which Michael acceptors are
Synthesis of Michael acceptor-based caged precursors through
Knoevenagel condensation
suited to serve in the design of caged precursors of fluorescent The one-step synthesis of Michael acceptors 4–10 was
unsymmetrical pyronins, depending on the specifications achieved from the known benzaldehyde derivative 2 and the
required by the targeted fluorescence-based bioanalysis or bio- corresponding C-nucleophile, using conditions previously
imaging application.
optimised for the preparation of dicyanomethylidene-based
probe 3 (i.e., cat. piperidine, anhydrous Na2SO4, EtOH).14
When this condensation reaction was performed at room
temperature, the formation of the desired product was only
observed with dimedone, 1,3-dimethylbarbituric acid and
Results and discussion
As briefly reminded above, our group has recently shown that Meldrum’s acid as C-nucleophiles. It is important to note that
proteases (i.e., PGA and LAP) are able to build the xanthene- the reaction with dimedone is less efficient with many side-
based fluorophore AR116 from mixed bis-aryl ether caged products being formed due to the great electrophilic reactivity
precursors (probes 1 and 2) and through a biocompatible of the resulting dimedone-based Michael acceptor 4. Heating
cyclisation/aromatisation process triggered by this biological under reflux was effective to provide the condensation
stimulus.10 However, the kinetics of pyronin formation adducts derived from less usual C-nucleophiles. However,
was too slow (over 10 h to achieve a significant level of under these latter conditions, 4-phenyl-1,3-dihydro-2H-1,5-
fluorescence) for considering the implementation of such benzodiazepin-2-one was found to be unreactive toward benz-
“covalent-assembly” type probes in diagnostic bioassays or in aldehyde 2 and we failed to obtain the claimed caged precur-
the most challenging context of in vivo fluorogenic imaging sor. Alternative conditions (i.e., the use of NaOAc as a base in
of disease-relevant enzymes. A considerable increase in the AcOH under reflux) previously reported for the Knoevenagel
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Scheme 2 Optimised synthesis of unsymmetrical pyronin AR116. (Boc2O = di-tert-butyl dicarbonate, FC (SiO2) = flash-column chromatography
over silica gel, O/N = overnight, RT = room temperature).
condensation between NSC 645039 and 4-(dimethylamino) required three successive purifications on C18-reversed phase
benzaldehyde23 were also tested but degradation of benz- silica (very poor isolated yield <2%).10 In addition, the recov-
aldehyde 2 and its cyclisation into the N-phenylacetyl deriva- ered fluorophore was still contaminated with a minor amount
tive of pyronin AR116 were observed. All PGA-sensitive (less than 10%) of the starting aldehyde. To circumvent these
“covalent-assembly” fluorogenic probes were isolated in mod- difficulties, a two-step synthetic route based on (1) Ullman-
erate to good yields (except for dimedone adduct 4) by conven- type coupling between N-Boc-3-iodoaniline 11 and 4-(diethyl-
tional flash-chromatography on silica gel. Optimal purity amino)salicylaldehyde and subsequent (2) TFA-mediated
(>95%) required for fluorescence measurements and enzy- cascade deprotection–cyclisation–dehydration reaction was
matic assays was readily achieved by further purification by devised (Scheme 2). As expected, the purification of AR116 was
semi-preparative RP-HPLC. Since compounds 4–6 and 10 were greatly facilitated and the isolated yield was dramatically
found to be poorly stable under aqueous acidic conditions (i.e., improved (14% over two steps).
0.1% aqueous formic acid and trifluoroacetic acid), ultrapure
water and MeCN were used as eluents. Conversely, 4,7-dihydroxy-
coumarin adduct 7 and hemicyanine-like derivatives 8 and 9 are
Comparative enzymatic activation of Michael acceptor-based
caged precursors
fully stable under aqueous acidic conditions and were recov- Before launching the campaign of fluorescence-based in vitro
ered as TFA salts. The mass percentages of TFA (17%, 16.5% assays with commercial PGA enzymes (from Escherichia coli),
and 19% respectively, corresponding to ca. one molecule of we have studied the photophysical properties of probes 4–10 in
TFA per molecule of probe) in freeze-dried samples were deter- phosphate buffer (100 mM, pH 7.6) containing less than 1% of
mined by ionic chromatography. All spectroscopic data (see DMSO originating from the dilution of 1.0 mg mL−1 stock
ESI‡), especially IR, NMR and mass spectrometry, were in solution in this latter solvent (see Fig. S1–S7‡ for the corres-
agreement with the structures assigned. Their purity was ponding spectral curves). All PGA-sensitive probes 4–10 exhibit
checked by RP-HPLC and found to be above 95% with the a strong electronic absorption either in the blue-cyan region
exception of Knoevenagel adducts poorly stable under aqueous (Abs λmax centered around 470–495 nm with ε in the range
acidic conditions (see ESI‡). Surprisingly, the Michael acceptor 32 300–29 500 M−1 cm−1 for probes 4–6, 8 and 10) or in the
moiety of the 4,7-dihydroxycoumarin adduct is sufficiently green-yellow spectral range for N-phenylacetyl rosamine 7 (Abs
electrophilic to undergo an intramolecular nucleophilic λmax = 507 and 552 nm, ε = 36 900 and 34 800 M−1 cm−1) and
addition of the adjacent phenylogous N-phenylacetylamine hemicyanine-like derivative 9 (Abs λmax = 548 nm, ε = 67 900
unit yielding unprecedented N-acyl rosamine dye 7 based on a M−1 cm−1) that contain a more extended conjugated π system.
pyronin–coumarin hybrid skeleton (Fig. 5). In addition to Unlike the malononitrile adduct 3, the excitation of these
these syntheses, we have also re-examined the preparation of probes did not produce detectable light emission (except for
unsymmetrical pyronin AR116 (used as a reference for in vitro Meldrum’s acid adduct 6 and hemicyanine-like derivative 9,
fluorescence assays and HPLC-fluorescence analyses) with the with weak emission centered at 612 and 593 nm respectively
aim of both improving the isolated yield and facilitating its and poor quantum yield determined at 1% or less, see the
purification. Indeed, Brønsted acid-mediated condensation of ESI‡ for detailed information) and the aggregation-induced
4-(diethylamino)salicylaldehyde with 3-aminophenol did not emission (AIE) phenomenon was not observed. The main con-
work well and the isolation of AR116 from the crude mixture sequence of this zero-background fluorescence for these caged
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Fig. 2 Time-dependent changes in the green-yellow fluorescence intensity (Ex./Em. 525/545 nm, slit 5 nm) of fluorogenic probes 2–10 (concen-
tration: 1.0 μM) in the presence of PGA (1 U) in PB (100 mM, pH 7.6) at 37 °C. Please note: PGA was added after 5 min of incubation of probe in PB
alone.
precursors is that the detection of PGA enzyme activity can be methylbarbituric acid and Meldrum’s acid and (2) lower electro-
achieved through an intensometric fluorogenic response.
All fluorogenic PGA assays and blank experiments were dicyanomethylidenyl moiety. Thus, the rate of the tandem cycli-
achieved through time-course measurements following sation–aromatisation process is negatively impacted. The posi-
philicity of the Michael acceptor compared to that of the
a
reliable protocol previously developed by us. The resulting tive counterpart of the relative lack of reactivity of this probe is
kinetic curves are shown in Fig. 2 (see Fig. S10–S16‡ for blank its full aqueous stability that is a key parameter of theranostic
curves). Since the quantitative conversion of the reference probe agents. The presence of free edaravone in the enzymatic reaction
(i.e., dicyanomethylidene-based probe 3) into pyronin AR116 mixture was unambiguously confirmed by HPLC-MS analysis
occurs within 30 min, this duration was chosen for all kinetics. carried out after a prolonged time of incubation (20 h, see Fig. 3
A rapid and gradual increase of fluorescence emission at and Fig. S38–S41‡). This product was identified through its
545 nm (Ex. 525 nm) was observed with three PGA-sensitive HPLC retention time (tR = 2.6 min, MS(ESI+): m/z = 175.5
Michael acceptor-based caged precursors 4–6. These results are [M + H]+, calcd for C10H11N2O+ 175.1) comparison and co-injec-
consistent with both our expectations and low pKa values of tion with commercial reference but also by MS analysis (full-
C-nucleophiles (acting as the best leaving groups) released scan and SIM modes). Interestingly, when the reaction with
through the 1,6-elimination process leading to xanthene aroma- PGA was conducted at a higher concentration (10 μM of probe
tisation (i.e., dimedone, 1,3-dimethylbarbituric acid and 10 vs. 1 μM for fluorescence-based assays), we also observed the
Meldrum’s acid). However, the level of fluorescence achieved is formation of a minor side-product (ca. 15%) identified as the
somewhat lower than that of the reference probe 3, particularly pyronin 13 substituted at the meso-position (i.e., C-9 position)
for the dimedone adduct (3200 AFU vs. 9200 AFU within by edaravone (tR = 4.0 min, MS(ESI+): m/z = 439.3 [M + H]+,
+
30 min) suggesting poor aqueous stability at physiological temp- calcd for C27H27N4O2 439.2 and UV-vis: λmax = 538 nm). This
erature of this “covalent-assembly” type probe. Indeed, the unexpected result confirms both the moderate electrophilic
nucleophilic addition of a water molecule (or hydroxide ion) to reactivity of the C-9 position of pyronins and nucleophilicity of
its activated double bond should lead to the release of dime- edaravone in its deprotonated form, under aqueous physiological
done and unveiling of the aldehyde functional group through a conditions. To the best of our knowledge, 10 is the first example
retro-Knoevenagel process. The propensity of probes 5 and 6 to of the “covalent-assembly” type probe whose enzymatic acti-
undergo such undesired hydrolysis is much more limited and vation leads to the simultaneous formation of a xanthene-based
this feature was further supported by comprehensive stability fluorophore and release of a bioactive substance. Indeed, the
studies (vide infra). Interestingly, a slower but still gradual only two examples of such fluorogenic prodrugs are nitroreduc-
increase of green-yellow fluorescence intensity was obtained tase (NTR)-sensitive caged precursors namely GMC-CAE-NO2
with the pyrazolone adduct 10 (2270 AFU within 30 min). This (Fig. 4A) and FDU-DB-NO2 (Fig. 4B) whose activation under
decrease of the kinetics of pyronin formation is closely related hypoxic conditions (and subsequent UV-irradiation for
to both the (1) higher pKa value of edaravone that may be GMC-CAE-NO2) produces a blue or green emitting 7-N,N-diethyl-
regarded as a less good leaving group than dimedone, 1,3-di- aminocoumarin derivative and a cytotoxic cancer chemotherapy
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Fig. 4 Hypoxia-activated anticancer theranostic prodrugs based on the
“covalent-assembly” strategy and already reported in the literature.24
The assumed PGA sensing mechanism for rosamine-based
pro-fluorophore 7 is dramatically different and based on the
more conventional enzyme-mediated aniline deprotection
process.1a,26 Since the released pyronin–coumarin hybrid
dye has never been reported in the literature, its photo-
physical properties and ability to display an internal energy
transfer process27 or PeT quenching28 are not known.
Therefore, the kinetic fluorescence measurements of PGA
activation at Ex./Em. 525/545 nm did not show a detectable
“turn-on” response (Fig. 1). In order to identify the primary
factor behind this negative outcome (i.e., wrong detection
parameters or poor substrate of PGA), fluorescence emission
and excitation spectra in the wavelength ranges anticipated
for these coumarin and pyronin chromophores have been
recorded after 30 min incubation of probe 7 with PGA (see
Fig. S13‡). The shape and position of the emission band
(centered at 560 nm) are consistent with a xanthene dye.
The excitation spectrum of the emissive species causing
this yellow fluorescence displays a single band centered at
535 nm. This indicates no direct π-conjugation in the ground
state between these two chromophores that take up a per-
pendicular geometry to each other, and no energy transfer
between them. We have therefore performed a second kinetic
experiment with the optimized set of Ex./Em. 535/560 nm
parameters and the expected fluorogenic “turn-on” response
was observed (190 AFU within 30 min, Fig. 5). However, the
low level of fluorescence reached suggests a poor quantum
yield for the released rosamine 14 that can be explained in
part by a tautomeric equilibrium with a weakly (or non-) fluo-
rescent species (Fig. 5).29
Fig. 3 RP-HPLC elution profiles (UV-vis and ESI+ mass detection,
system B) of the enzymatic reaction mixture of probe 10 with PGA (20 h
of incubation in PB at 37 °C). (A) UV detection at 260 nm, (B) visible
detection at 525 nm, (C) ESI+ mass detection in the SIM mode, (D) ESI+
mass spectrum of the released edaravone and (E) ESI+ mass spectrum
of pyronin–edaravone adduct 13.
agent (gemcitabine and floxuridine respectively) for bioimaging,
This comparative study concludes with reactions between
tracking drug release and anticancer application.24 In this the PGA enzyme and hemicyanine-like derivatives 8 and 9
context, a slow liberation of drug molecules may be advantageous and for which no significant increase in pyronin fluo-
to reduce the “burst effect” which is the primary source of severe rescence within the time range of 30 min was observed.
side-effects associated with such therapeutic treatments.25
Theoretically, two possible interpretations can be put forth
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Fig. 6 UV-vis absorbance changes (at λmax) of fluorogenic probes 3, 5,
6, 8, 9 and 10 after 30 min incubation in three distinct aqueous buffers
(PB, 100 mM, pH 7.6 and borate buffer, 100 mM, pH 8.6 and 9.5) at
25 °C (concentration: 2.0 μM).
Fig. 5 Fluorescence-based PGA assay with pyronin–coumarin hybrid
pro-fluorophore 7. (Top) Activation mechanism based on the de-
protection of fluorogenic primary aniline and possible tautomeric equili-
brium, (bottom) time-dependent changes in the yellow fluorescence
Since this probe exhibits the highest ability to absorb
intensity (Ex./Em. 535/560 nm, slit 5 nm) of fluorogenic probe 7 (con- visible light (vide supra), we assume that the photooxidative
centration: 1.0 μM) in the presence of PGA (1 U) in PB (100 mM, pH 7.6)
at 37 °C. Please note: PGA was added after 5 min of incubation of probe
in PB alone.
N-dealkylation process leading to photobluing of fluorescent
organic dyes bearing (di)alkylamino auxochromic groups,31
such as pyronin AR116, occurs upon prolonged illumination at
525 nm implemented during the fluorescence-based in vitro
assays.
to explain these results: (1) undesired conversion of the
hemicyanine-based Michael acceptor moiety into a less reac-
tive aldehyde functionality through the retro-Knoevenagel
reaction, known to be favoured in basic aqueous media and
Aqueous stability and thiol-reactivity of Michael
acceptor-based caged precursors
already reported for some cyanine dyes30 and/or the (2) poor Since the undesired reaction of Michael acceptor-based caged
leaving group ability of the N-quaternised aza-heterocycle precursors with hydroxide ions, water or biological nucleo-
bearing an activated methyl group that directly affects the philes would lead either to adducts not prone to cyclisation–
rate of the 1,6-elimination process. Since, we did not observe elimination to yield the pyronin or to the premature release of
any change (i.e., blue-shift of the absorption maximum and the second molecule of interest (e.g., drugs such as barbitu-
the hypochromic effect in line with the loss of an extended π rates or edaravone) through the retro-Knoevenagel reaction, it
system) in the UV-vis absorption spectra of 8 and 9 during was essential to conduct further studies to assess the pH-
their incubation in phosphate buffer (see bar charts shown dependent stability of these probes and their possible reaction
in Fig. 6), the second hypothesis is preferred. Unfortunately, with biothiols. On the basis of the findings of in vitro enzy-
the pKa values of active methyl compounds 1,4-dimethyl- matic assays, the most promising probes 3, 5, 6 and 10 have
pyridinium and 1-ethyl-2,3,3-trimethylindolenium (iodide been selected and subjected to incubation in three different
salts) are not available to further support our hypothesis. All aqueous buffers (i.e., phosphate pH 7.6, borate pH 8.5 and 9.5).
the same, we may conclude that N-quaternised aza-hetero- Monitoring of absorbance at their maximum wavelength (in
cycles bearing an activated methyl group are not suitable the range of 445–490 nm) over time, which is assumed to be
candidates to promote the activation kinetics of pyronin lost upon the nucleophilic addition of a water molecule or
caged precursors.
hydroxide ion and possibly retro-Knoevenagel reaction leading
The in situ formation of pyronin AR116 in samples from to conversion to the corresponding aldehyde (bathochromic
enzymatic fluorescence assays was further confirmed by shift), is a simple way to rapidly check the overall stability of
RP-HPLC (coupled with fluorescence detection) analyses, these Michael acceptor-based caged precursors. It could be
including the comparison of the observed retention time (tR
=
seen that there were negligible absorbance changes for probes
3.8 min) with that of an authentic sample of synthetic pyronin 3, 6 and 10 at all pH values (see bar charts shown in Fig. 6),
AR116 used as a reference (see Fig. S21–S37‡ for the RP-HPLC indicating their good aqueous stability. Conversely, a signifi-
elution profiles). By analogy with our previous observations cant decrease in absorbance at 490 nm was observed for the
made during fluorescent enzyme assays with aldehyde-based probe bearing a methylidene 1,3-dimethylbarbituric acid portion,
“covalent-assembly” probe 2, a second fluorescent species upon an increase of pH from 7.6 to 9.5. For practically equi-
identified as mono-dealkylated pyronin was observed but only valent performance in fluorogenic “turn-on” response, it
on the RP-HPLC-fluorescence elution profile (tR = 3.5 min) of would then be preferable to use a dicyanomethylidenyl or
the crude enzymatic reaction of hemicyanine-like derivative 9. methylidene Meldrum’s acid moiety rather than the more reac-
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Fig. 7 Thiol reactivity of barbiturate-based probe 5 in PB (100 mM, pH 7.6). (A) Time-dependent changes in the green-yellow fluorescence intensity
(Ex./Em. 525/545 nm, slit 5 nm) of 5 (concentration: 1.0 μM) in the presence of PGA (1 U) with or without glutathione (GSH = R-SH, 50 equiv.) at
37 °C. Please note: PGA was added after 5 min of incubation of probe in PB alone. (B) Time-dependent changes in absorbance (490 nm) of 5 (con-
centration: 2.0 μM) after sequential addition of GSH (50 equiv.) and NEM (50 equiv.) at 25 °C. (GSH = glutathione, NEM = N-ethylmaleimide).
tive Michael acceptor namely methylidene 1,3-dimethyl- has led to the resurgence of the visible absorbance feature of 5
barbituric acid.
(Fig. 7B). We can conclude that thiols may be protective for
The other important feature for considering the use of such Michael acceptor-based caged precursors by preventing their
probes in complex biological media is their chemical inertness hydration and subsequent conversion to less reactive aldehyde
toward biothiols such as glutathione (GSH) whose concen- derivative 2 through the retro-Knoevenagel reaction. This
tration could reach high values, especially in tumor cells feature is particularly valuable for biomedical applications in
(0.5–10 mM).32 In this context, further fluorescence PGA assays living systems.
and absorbance measurements were performed in phosphate
buffer (pH 7.6) and in the presence of 50 equiv. of GSH.
Gratifyingly, no deleterious effect of GSH on the fluorogenic
“turn-on” response and hence on the in situ pyronin formation
Conclusions
process was observed (see Fig. 7A and Fig. S17–S19‡). Perhaps In summary, a significant advance has been made both to fine-
more surprising are the results obtained with barbiturate- tune the reactivity and optimise the properties of “covalent-
based probe 5. Indeed, a rapid and dramatic decrease of its assembly” type probes that utilise the targeted enzyme to build
absorption at 490 nm was observed, confirming that the thiol- a detectable pyronin fluorophore under physiological con-
Michael addition reaction occurred (see Fig. 7B and bar charts ditions. Indeed, the change of the Michael acceptor moiety
shown in Fig. S20‡). However, the apparent disappearance of involved in the domino cyclisation–aromatisation reaction
the Michael acceptor moiety is not likely to negatively impact leading to a xanthene scaffold was identified as a subtle but
the tandem cyclisation–aromatisation process yielding
a
effective structural modification to dramatically impact the
pyronin structure, because the level of fluorescence achieved kinetics of this unusual protease-triggered fluorogenic process.
after 30 min incubation of probe 5 with PGA and GSH is Indeed, in the context of our fluorescence-based bioassay
higher than that obtained with the enzymatic reaction con- format, Michael acceptor adducts derived from C-nucleophiles,
ducted without this thiol additive (Fig. 7A). This surprising namely 1,3-dimethylbarbituric acid, malononitrile and
result can be potentially explained by the reversibility of the Meldrum’s acid, are quantitatively converted into pyronin
GSH-probe adduct, demonstrated by a further experiment in AR116 within 30 min of incubation with PGA at physiological
which the probe 5 was sequentially incubated with GSH pH. The enhanced intensity and shortened time-scale of the
(50 equiv.) and N-ethylmaleimide (NEM, 50 equiv.).33 Indeed, resulting fluorogenic “turn-on” response are first steps toward
the addition of an equimolar amount of this thiol scavenger the future implementation of such a “covalent-assembly” strat-
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egy towards spatio-temporal profiling of disease-relevant
enzymes in live cells and in vivo. We also demonstrated that the
Experimental section
apparent discrepancy between the reactivity and aqueous stabi- For all experimental details related to photophysical character-
lity of these probes related to the enhanced electrophilicity of isation, fluorescence-based in vitro assays and HPLC-fluo-
their Michael acceptor moiety could be dismissed out of hand rescence/-MS analyses, see the ESI.‡
by the protective effect of biothiols demonstrated through in vitro
General
assays conducted with GSH. This trick cannot, however, be used
for the dimedone-based “covalent-assembly” type probe 4 Unless otherwise noted, all commercially available reagents
because of its marked chemical instability. The high electrophili- and solvents were used without further purification. TLC was
city of the corresponding Michael acceptor and the good leaving carried out on Merck DC Kieselgel 60 F-254 aluminum sheets.
group ability of dimedone are insurmountable obstacles to the The spots were directly visualised or through illumination with
further use of this moiety as an effective promoter of activation a UV lamp (λ = 254/365 nm) and/or staining with KMnO4 solu-
kinetics of this unusual class of enzyme-responsive fluorogenic tion. Purifications by flash column chromatography were per-
probes. Interestingly, the in situ formation of fluorescent formed on silica gel (40–63 µm) from VWR. Anhydrous DMSO
pyronin AR116 at a slower rate was achieved with a pyrazolone- was purchased from Carlo Erba, and stored over 3 Å molecular
based Michael acceptor 10 (i.e., edaravone as a C-nucleophile). sieves. Absolute EtOH (+99.8%, Reag. Ph. Eur. for analyses)
In this latter case, pyronin formation is accompanied by the was purchased from VWR. Piperidine (peptide grade, SOL-010)
release of edaravone as demonstrated by HPLC-MS analyses. and PGA (from Escherichia coli, EZ50150, 841 U mL−1) were
This strategy could provide a novel and promising platform for provided by Iris Biotech GmbH. Formic acid (FA, puriss p.a.,
the facile construction of theranostic prodrugs activated by ACS reagent, reag. Ph. Eur., ≥98%), 4,7-dihydroxycoumarin
enzymes or reactive bioanalytes associated with a specific (97%) and DMSO (molecular biology grade) were provided by
disease.13 Indeed, the versatile synthetic route toward mixed bis- Sigma-Aldrich. Edaravone, N-ethylmaleimide (NEM) and gluta-
aryl ether derivatives devised by us could be used by changing thione (reduced form, GSH, 98%) were purchased from Rhone-
only the analyte-sensitive trigger group and C-nucleophile. Poulenc Rorer, Pierce and Acros respectively. The HPLC-gradi-
However, we are fully aware that the bioactive molecule chosen ent grade acetonitrile (MeCN) was obtained from Carlo Erba or
for the construction of such fluorogenic theranostic conjugates VWR. All aqueous buffers used in this work and aqueous
must bear an easily enolisable position or be functionalised mobile-phases for HPLC were prepared using water purified
with a C-nucleophile having low pKa and that does not nega- with
a PURELAB Ultra system from ELGA (purified to
tively affect its biological properties. Moreover, Knoevenagel con- 18.2 MΩ cm). Aldehyde- and dicyanomethylidene-based PGA
densation with fluorescent organic dyes bearing an enolisable probes [2097130–07–7] 2 and [2305970–99–2] 3, 1,4-dimethyl-
carbon should enable the rapid development of two-channel pyridinium iodide [2301–80–6] and 1-ethyl-2,3,3-trimethyl-
fluorescent probes with a more sophisticated signaling mecha- indolenium iodide [14134–81–7] were prepared according to
nism,34 especially for the simultaneous detection of two distinct the literature procedures.8w,10,14,37
(bio)analytes.6a,35 For such a purpose, a possible and straight-
forward strategy would be to perform Knoevenagel condensation
Instruments and methods
with a cyclic C-nucleophile that contains an additional group Freeze-drying operation was performed with a Christ Alpha
(e.g., amino or azido group, carboxylic acid, terminal alkyne, 2–4 LD plus. Centrifugation steps were performed with a
etc.)36 suitable for its covalent conjugation to a pro-fluorophore Thermo Scientific Espresso Personal Microcentrifuge instru-
sensitive to the second targeted (bio)analyte (Fig. 8).
ment. 1H-, 13C- and 19F-NMR spectra were recorded either on a
Bruker Avance III 500 MHz or on a Bruker Avance III HD
600 MHz spectrometer (equipped with double resonance
broad band probes). Chemical shifts are expressed in parts per
million (ppm) from the residual non-deuterated solvent
signal.38 J values are expressed in Hz. IR spectra were recorded
with a Bruker Alpha FT-IR spectrometer equipped with a uni-
versal ATR sampling accessory. The bond vibration frequencies
are expressed in reciprocal centimeters (cm−1). HPLC-MS ana-
lyses were performed on a Thermo-Dionex Ultimate 3000
instrument (pump + autosampler at 20 °C + column oven at
25 °C) equipped with a diode array detector (Thermo-Dionex
DAD 3000-RS) and an MSQ Plus single quadrupole mass
spectrometer. HPLC-fluorescence analyses were performed
with the same instrument coupled to a RS fluorescence detec-
tor (Thermo-Dionex, FLD 3400-RS). Purifications by semi-pre-
parative HPLC were performed on a Thermo-Dionex Ultimate
3000 instrument (semi-preparative pump HPG-3200BX)
Fig. 8 A possible strategy toward double-emission fluorescent probe
for discriminatory detection of two distinct analytes, based on the con-
jugation of a “covalent-assembly” type probe to a conventional phenol-
based pro-fluorophore (e.g., 7-hydroxycoumarin-4-acetic acid) through
a functionalised cyclic C-nucleophile.
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equipped with an RS Variable Detector (VWD-3400RS, four dis- the same eluents and gradient as system A. Fluorescence
tinct wavelengths within the range 190–900 nm). Ion chrom- detection was achieved at 45 °C at the following Ex./Em.
atography analyses (for TFA quantification) were performed channels: 525/545 nm, 510/530 nm and 440/600 nm (sensi-
using a Thermo Scientific Dionex ICS 5000 ion chromatograph tivity: 1, PMT 1, filter wheel: auto).
equipped with a conductivity detector CD (Thermo Scientific
General procedure for the synthesis of Michael acceptor-
Dionex) and a conductivity suppressor ASRS-ultra II 4 mm based caged precursors. To a stirred solution of aldehyde 2
(Thermo Scientific Dionex). Low-resolution mass spectra (40 mg, 0.1 mmol, 1 equiv.) in absolute EtOH (5 mL),
(LRMS) were recorded on a Thermo Scientific MSQ Plus single C-nucleophile (0.105 mmol, 1.05 equiv.), anhydrous Na2SO4
quadrupole equipped with an electrospray (ESI) source (direct (10 mg) and piperidine (1 drop) were successively added. The
introduction or LC-MS coupling). High-resolution mass resulting reaction mixture was stirred for a defined duration
spectra (HRMS) were recorded on a Thermo LTQ Orbitrap XL and temperature depending on the C-nucleophile used. After
apparatus equipped with an ESI source.
completion of the reaction, the mixture was evaporated under
reduced pressure and the resulting residue was directly puri-
fied by flash-column chromatography over silica gel (ca.
High-performance liquid chromatography separations
Several chromatographic systems were used for the analytical 12 g). Further purification by semi-preparative RP-HPLC was
experiments (HPLC-MS or HPLC-fluorescence) and the purifi- achieved to obtain the desired PGA-sensitive probe with a
cation steps: System A: RP-HPLC-MS (Phenomenex Kinetex C18 purity >95% required for fluorescence measurements and
column, 2.6 μm, 2.1 × 50 mm) with MeCN (+0.1% FA) and 0.1% enzymatic assays.
aqueous formic acid (aqueous FA, pH 2.7) as eluents [5% MeCN
Dimedone-based PGA-sensitive probe (4). Dimedone was
(0.1 min) followed by linear gradient from 5% to 100% (5 min) used as a C-nucleophile (14.7 mg, 0.105 mmol, 1.05 equiv.).
of MeCN] at a flow rate of 0.5 mL min−1. UV-visible detection The reaction mixture was stirred at RT overnight and under
was achieved at 220, 260, 450 and 500 nm (+diode array detec- reflux for 1 h. The crude product was purified by flash-column
tion in the range of 220–700 nm). Low resolution ESI-MS detec- chromatography (step gradient of EtOAc in heptane from
tion in the positive/negative mode (full scan, 100–1000 a.m.u., 40% to 100%) and semi-preparative RP-HPLC (system F, tR
=
data type: centroid, needle voltage: 3.0 kV, probe temperature: 37.5–39.5 min). The desired PGA-sensitive probe 4 was recov-
350 °C, cone voltage: 75 V and scan time: 1 s). System B: system ered (after freeze-drying) as a red amorphous powder (ca.
A with UV-visible detection at 220, 260, 470 and 525 nm 0.1 mg, <1 μmol, yield <1%). Please note: This compound was
(+diode array detection in the range of 220–800 nm). Low found to be too unstable to be isolated in significant amounts
1
resolution ESI-MS detection in the positive/negative mode (full required for H and 13C NMR analyses. IR (ATR): ν = 2923, 2853,
scan, 100–1000 a.m.u. and SIM mode with the following mass 2319, 2221, 2197, 2176, 2161, 2073, 2049, 2038, 2003, 1991,
range (m/z 175.5 0.5)). System C: system A with ultrapure H2O 1974, 1736, 1707, 1611, 1508, 1458, 1377, 1350, 1260, 1111,
and MeCN (without FA additive) as eluents. System D: semi- 1027, 796, 669 cm−1; please note: partial degradation of the
preparative RP-HPLC (SiliCycle SiliaChrom C18 column, 10 μm, product was noted during the RP-HPLC analysis conducted with
20 × 250 mm) with MeCN and ultrapure H2O as eluents [25% or without the FA additive. HPLC (system C): tR = 5.6 min; LRMS
MeCN (5 min), followed by a gradient of 25% to 55% MeCN (ESI+, recorded during RP-HPLC analysis): m/z 525.4 [M + H]+
+
(10 min), then 55% to 100% MeCN (45 min)] at a flow rate of (100), calcd for C33H37N2O4 525.3; LRMS (ESI−, recorded
20.0 mL min−1. Quadruple UV-vis detection was achieved at during RP-HPLC analysis): m/z 523.2 [M − H]− (100), calcd for
220, 260, 460 and 550 nm. System E: system D with UV-visible C33H35N2O4 523.3; HRMS (ESI+): m/z 525.27370 [M + H]+,
−
detection at 220, 260, 470 and 530 nm. System F: system D with calcd for C33H37N2O4 525.27478, and 547.25662 [M + Na]+,
+
UV-visible detection at 220, 260, 280 and 475 nm. System G: calcd for C33H36N2O4Na+ 547.25673; UV-vis: λmax (PB)/nm 281
system D with the following gradient [30% MeCN (5 min), fol- and 495 (ε/dm3 mol−1 cm−1 20 700 and 11 900).
lowed by a gradient of 30% to 60% MeCN (10 min), then 60%
to 100% MeCN (40 min)] at a flow rate of 20.0 mL min−1
Barbiturate-based
Dimethylbarbituric acid was used as a C-nucleophile (16.4 mg,
PGA-sensitive
probe
(5).
1,3-
.
Quadruple UV-vis detection was achieved at 220, 260, 350 and 0.105 mmol, 1.05 equiv.). The reaction mixture was stirred at
470 nm. System H: semi-preparative RP-HPLC (SiliCycle RT overnight. The crude product was purified by flash-column
SiliaChrom C18 column, 10 μm, 20 × 250 mm) with MeCN and chromatography (step gradient of MeOH in DCM from 0%
aqueous 0.1% TFA (pH 2.0) as eluents [10% MeCN (5 min), fol- to 20%) and semi-preparative RP-HPLC (system E, tR
lowed by a gradient of 10% to 30% MeCN (10 min), then 30% 32.0–35.0 min). The desired PGA-sensitive probe 5 was recov-
to 100% MeCN (95 min)] at a flow rate of 20.0 mL min−1
ered (after freeze-drying) as an orange amorphous powder
=
.
Quadruple UV-vis detection was achieved at 220, 260, 500 and (30.9 mg, 57 μmol, yield 57%). IR (ATR): ν = 3253, 2971, 2066,
550 nm. System I: system H with the following gradient [25% 1710, 1650, 1609, 1527, 1501, 1419, 1387, 1371, 1342, 1306,
MeCN (5 min), followed by a gradient of 25% to 45% MeCN 1266, 1196, 1169, 1072, 968, 872, 824, 786, 756, 708, 679,
1
(10 min), then 45% to 100% MeCN (75 min)] at a flow rate of 653 cm−1; H NMR (500 MHz, CDCl3): δ = 9.09–8.83 (m, 2 H),
20.0 mL min−1. Quadruple UV-vis detection was achieved at 7.42–7.35 (m, 3 H), 7.34–7.29 (m, 3 H), 7.26–7.18 (m, 2 H), 7.12
220, 260, 350 and 550 nm. System J: RP-HPLC-fluorescence (d, J = 2.2 Hz, 1 H), 6.75–6.67 (m, 1 H), 6.45 (dd, J = 9.6, J =
(Phenomenex Kinetex C18 column, 2.6 μm, 2.1 × 50 mm) with 2.7 Hz, 1 H), 5.97 (d, J = 2.7 Hz, 1 H), 3.70 (s, 2 H), 3.39
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(s, 3 H), 3.37–3.30 (m, 7 H), 1.12 (t, J = 7.1 Hz, 6 H) ppm; 8.32 (d, J = 2.0 Hz, 1 H), 7.93 (d, J = 9.1 Hz, 1 H), 7.81 (d, J =
13C NMR (126 MHz, CDCl3): δ = 169.2, 164.3, 162.9, 161.9, 9.7 Hz, 1 H), 7.70 (d, J = 8.5 Hz, 1 H), 7.43 (dd, J = 9.1 Hz, J =
156.7, 154.7, 152.1, 151.8, 139.4, 137.3, 134.5, 130.2, 129.6, 2.0 Hz, 1 H), 7.40–7.31 (m, 3 H), 7.31–7.21 (m, 2 H), 7.05 (d,
129.4, 127.8, 115.6, 115.3, 112.7, 111.3, 109.3, 106.8, 99.4, 45.2, J = 2.4 Hz, 1 H), 6.66 (dd, J = 8.4 Hz, J = 2.4 Hz, 1 H), 6.58 (t, J =
45.0, 28.9, 28.37, 12.8 ppm; please note: partial degradation of 2.9 Hz, 1 H), 3.78 (s, 2 H), 3.72 (q, J = 7.1 Hz, 4 H), 1.54–0.96
the product was noted during the RP-HPLC analysis conducted (m, 6 H) ppm; 13C NMR (151 MHz, DMSO-d6): δ = 171.1, 171.0,
with or without the FA additive. HPLC (system C): tR = 5.4 min 162.4, 161.4, 158.9, 157.2, 156.1, 155.3, 146.6, 135.6, 135.3,
(purity 74% at 260 nm, 90% at 450 nm and 76% at 500 nm); 132.7, 129.8, 128.9, 127.2, 118.1, 117.7, 117.3, 115.9, 111.7,
LRMS (ESI+, recorded during RP-HPLC analysis): m/z 541.3 [M 104.8, 102.0, 101.9, 95.7, 92.2, 46.1, 43.9, 43.8 ppm; 19F NMR
+
+ H]+ (100), calcd for C31H33N4O5 541.2; LRMS (ESI−, (565 MHz, DMSO-d6): δ = −73.5 (s, 3 F, CF3-TFA) ppm; HPLC
recorded during RP-HPLC analysis): m/z 539.0 [M − H]− (100), (system A): tR = 4.2 min (purity >99% at 260 nm, >99% at
−
calcd for C31H31N4O5 539.2; HRMS (ESI+) m/z 541.24545 450 nm and >99% at 500 nm); LRMS (ESI+, recorded during
+
[M + H]+, calcd for C31H33N4O5 541.24455, and 563.22691 RP-HPLC analysis): m/z 561.3 [M + H]+ (100), calcd for
[M + Na]+, calcd for C31H32N2O6Na+ 563.22649; UV-vis: λmax C34H29N2O6 561.2; LRMS (ESI−, recorded during RP-HPLC
+
(PB)/nm 251 and 493 (ε/dm3 mol−1 cm−1 29 800 and 31 600).
analysis): m/z 559.1 [M − H]− (100), calcd for C34H27N2O6
−
Meldrum’s acid-based PGA-sensitive probe (6). Meldrum’s 559.2; HRMS (ESI+): m/z 561.20278 [M + H]+, calcd for
acid was used as a C-nucleophile (15.1 mg, 0.105 mmol, 1.05 C34H29N3O6 561.20201, and 583.18333 [M + Na]+, calcd for
+
equiv.). The reaction mixture was stirred at RT for 5 h. The C34H28N3O6Na+ 583.18396; UV-vis: λmax (PB)/nm 303, 507 and
crude product was purified by flash-column chromatography 552 (ε/dm3 mol−1 cm−1 39 000, 36 900 and 34 800).
(step gradient of MeOH in DCM from 0% to 10%) and semi-
Hemicyanine-based
PGA-sensitive
probe
(8).
1,4-
preparative RP-HPLC (system D, tR = 32.0–33.5 min). The Dimethylpyridinium iodide was used as a C-nucleophile
desired PGA-sensitive probe 6 was recovered as a red amor- (24.7 mg, 0.105 mmol, 1.05 equiv.). The reaction mixture was
phous powder (25.2 mg, 47 μmol, yield 47%). IR (ATR): ν = stirred under reflux for 6 h. The crude product was purified by
3315, 3087, 2976, 2933, 1685, 1602, 1542, 1499, 1436, 1376, flash-column chromatography (eluent: DCM–EtOAc, 8 : 2 (v/v)
1346, 1297, 1256, 1177, 1121, 1095, 1075, 1003, 958, 932, 892, then DCM–MeOH, 95 : 5, v/v) and semi-preparative RP-HPLC
826, 789, 757, 722, 695, 684, 645 cm−1 1H NMR (500 MHz, (system H, tR = 36.0–43.0 min). TFA salt of the desired PGA-
;
CDCl3): δ = 9.26 (d, J = 1.9 Hz, 1 H), 9.11 (dd, J = 9.5 Hz, J = 1.9 sensitive probe 8 was recovered (after freeze-drying) as a red
Hz, 1 H), 7.99 (s, 1 H), 7.77–7.65 (m, 5 H), 7.65–7.56 (m, 3 H), amorphous powder (22.5 mg, 37 μmol, yield 37%). IR (ATR):
7.06 (d, J = 7.5 Hz, 1 H), 6.90–6.65 (m, 1 H), 6.26 (t, J = 2.2 Hz, ν = 3259, 3061, 2973, 2931, 2630, 2067, 1670, 1645, 1573, 1518,
1 H), 4.06 (d, J = 2.5 Hz, 2 H), 3.67 (q, J = 7.3 Hz, 4 H), 2.12 (t, 1418, 1481, 1454, 1436, 1404, 1378, 1353, 1340, 1305, 1272,
J = 1.5 Hz, 6 H), 1.46 (td, J = 7.3 Hz, J = 4.0 Hz, 6H) ppm; 1238, 1226, 1177, 1124, 1093, 1075, 1043, 961, 871, 821, 796,
13C NMR (126 MHz, CDCl3): δ = 169.4, 165.6, 165.6, 163.0, 717, 695, 685 cm−1 1H NMR (500 MHz, CDCl3): δ = 9.46 (s,
;
156.1, 156.0, 154.8, 150.9, 139.6, 136.6, 134.6, 130.1, 129.6, 1 H), 8.26–7.96 (m, 2 H), 7.61 (d, J = 15.9 Hz, 1 H), 7.46 (s,
129.2, 127.7, 115.8, 115.6, 111.7, 111.6, 107.2, 103.5, 98.7, 98.6, 1 H), 7.40 (d, J = 8.9 Hz, 1 H), 7.34 (s, 2 H), 7.23 (d, J = 6.9 Hz,
45.2, 44.8, 27.4, 12.7 ppm; HPLC (system A): tR = 5.4 min 3 H), 7.15 (t, J = 7.4 Hz, 2 H), 7.08 (q, J = 8.6 Hz, J = 8.1 Hz,
(purity 95% at 260 nm, 99% at 450 nm and 85% at 500 nm); 2H), 6.65 (d, J = 15.9 Hz, 1 H), 6.53 (d, J = 8.1 Hz, 1 H), 6.35 (d,
LRMS (ESI+, recorded during RP-HPLC analysis): m/z 529.2 [M J = 8.8 Hz, 1 H), 6.00 (s, 1 H), 3.88 (s, 3 H), 3.63 (s, 2 H), 3.20
+ H]+ (100), calcd for C31H33N2O6 559.2; LRMS (ESI−, (q, J = 7.1 Hz, 4 H), 1.01 (t, J = 7.1 Hz, 6 H) ppm; 13C NMR
+
recorded during RP-HPLC analysis) m/z 527.3 [M − H]− (100), (126 MHz, CDCl3): δ = 170.5, 158.4, 157.2, 154.6, 151.7, 143.4,
−
calcd for C31H31N2O6 557.2; HRMS (ESI+) m/z 529.23410 140.6, 137.4, 135.6, 130.4, 130.0, 129.6, 128.7, 127.1, 122.3,
[M + H]+, calcd for C31H33N2O6 559.23331, and 551.21596 116.5, 115.5, 113.5, 113.4, 110.8, 108.3, 101.7, 46.6, 44.9, 44.2,
+
[M + Na]+, calcd for C31H33N2O6Na+ 551.21526; UV-vis: λmax 12.7, 1.2 ppm; 19F NMR (470 MHz, CDCl3): δ = −75.3 (s, 3 F,
(PB)/nm 254, 286, 487 (ε/dm3 mol−1 cm−1 23 000, 15 100 and CF3–TFA) ppm; HPLC (system A): tR = 4.5 min (purity >99% at
29 500); fluorescence λmax (PB)/nm 612 (ΦF 1%).
Rosamine-based PGA-sensitive probe
260 nm, >99% at 450 nm and 100% at 500 nm); LRMS (ESI+,
(7).
4,7- recorded during RP-HPLC analysis): m/z 492.3 [M]+° (100),
+
Dihydroxycoumarin was used as a C-nucleophile (18.7 mg, calcd for C32H33N3O2 492.3; LRMS (ESI−, recorded during
0.105 mmol, 1.05 equiv.). The reaction mixture was stirred RP-HPLC analysis): m/z 536.0 [M + FA − 2H]− (40), calcd for
−
under reflux for 3 h. The crude product was purified by flash- C33H34N3O4 536.3, and 582.2 [M + 2FA − 2H]− (100), calcd
column chromatography (step gradient of MeOH in DCM from for C34H36N3O6 582.3; HRMS (ESI+): m/z 492.26337 [M]+°,
−
+
0% to 10%) and semi-preparative RP-HPLC (system H, tR
=
calcd for C32H34N3O2 492.26455; UV-vis: λmax (PB)/nm 469
38.0–40.0 min). TFA salt of the desired PGA-sensitive probe 7 (ε/dm3 mol−1 cm−1 32 300).
was recovered (after freeze-drying) as a dark purple amorphous
Hemicyanine-based PGA-sensitive probe (9). 1-Ethyl-2,3,3-tri-
powder (7.0 mg, 10 μmol, yield 10%). IR (ATR): ν = 2981, 1673, methylindolenium iodide was used as a C-nucleophile (33 mg,
1641, 1589, 1506, 1451, 1346, 1308, 1264, 1235, 1195, 1159, 0.105 mmol, 1.05 equiv.). The reaction mixture was stirred
1128, 1073, 1007, 989, 948, 824, 798, 772, 749, 704, 652 cm−1
;
under reflux for 3 h. The crude product was purified by flash-
1H NMR (600 MHz, DMSO-d6): δ = 11.02 (s, 1 H), 10.14 (s, 1 H), column chromatography (step gradient of MeOH in DCM from
8928 | Org. Biomol. Chem., 2019, 17, 8918–8932
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Paper
H]+, calcd for C35H35N4O3
+
0% to 10%) and semi-preparative RP-HPLC (system I, tR
=
(ESI+) m/z 559.27091 [M
+
29.0–38.0 min). TFA salt of the desired PGA-sensitive probe 9 559.27037, 581.25261 [M + Na]+, calcd for C35H34N4O3Na+
was recovered (after freeze-drying) as a purple amorphous 581.25231; UV-vis: λmax (PB)/nm 257 and 486 (ε/dm3 mol−1
powder (39.6 mg, 58 μmol, yield 58%). IR (ATR): ν = 3249, cm−1 29 500 and 29 700).
3193, 3059, 3026, 2972, 2926, 2872, 2363, 2163, 2147, 2114,
N-Boc-3-iodoaniline [143390–49–2] (11). 3-Iodoaniline (2 g,
2036, 1991, 1792, 1683, 1601, 1567, 1517, 1466, 1410, 1399, 9.13 mmol, 1 equiv.) was dissolved in absolute EtOH (44 mL)
1373, 1352, 1320, 1300, 1256, 1215, 1192, 1170, 1157, 1115, and kept under an argon atmosphere. Boc2O (2 g, 9.13 mmol,
1071, 1044, 1016, 981, 947, 926, 874, 821, 796, 757, 708, 694, 1 equiv.) and TEA (2.54 mL, 18.26 mmol, 2 equiv.) were succes-
1
681 cm−1; H NMR (500 MHz, CDCl3): δ = 10.84 (d, J = 5.8 Hz, sively added and the resulting reaction mixture was stirred at
1 H), 8.34 (d, J = 15.2 Hz, 1 H), 7.90–7.73 (m, 2 H), 7.62 (dd, J = RT overnight. The reaction was checked for completion by TLC
8.1 Hz, J = 1.9 Hz, 1 H), 7.52–7.32 (m, 5 H), 7.31–7.18 (m, 4 H), (eluent: DCM 100%) and the mixture was then concentrated
7.14 (dd, J = 8.4 Hz, J = 6.3 Hz, 1 H), 7.05 (d, J = 15.2 Hz, 1 H), under reduced pressure. The resulting residue was dissolved in
6.65 (dd, J = 8.1 Hz, J = 2.4 Hz, 1 H), 6.53 (dd, J = 9.3 Hz, J = 2.5 DCM (40 mL) and washed with aqueous 1.0 M HCl thrice. The
Hz, 1 H), 6.05 (d, J = 2.5 Hz, 1 H), 4.25 (q, J = 7.3 Hz, 2 H), 3.81 organic layer was dried over anhydrous MgSO4 and concen-
(s, 2 H), 3.34 (q, J = 7.2 Hz, 4 H), 1.59 (s, 6 H), 1.41 (t, J = 7.2 trated over reduced pressure to give N-Boc-3-iodoaniline 11 as
Hz, 3 H), 1.12 (t, J = 7.1 Hz, 6 H) ppm; 13C NMR (126 MHz, a brown oily solid (2.26 g, 7.12 mmol, yield 78%). This product
CDCl3): δ = 178.3, 171.1, 162.1, 161.2, 161.0, 155.6, 155.4, was directly used in the next step without further purification.
149.4, 142.1, 142.0, 140.7, 136.2, 129.9, 129.7, 129.2, 128.3, Rf (DCM): 0.80; 1H NMR (500 MHz, CDCl3): δ = 7.83 (t, J =
127.5, 126.5, 122.6, 118.8, 116.7, 116.4, 114.1, 113.2, 112.2, 1.8 Hz, 1 H), 7.35 (ddd, J = 7.9 Hz, J = 1.8 Hz, J = 1.0 Hz, 1 H),
111.2, 109.3, 103.1, 100.4, 50.8, 45.6, 44.0, 40.8, 27.8, 12.8, 7.33–7.22 (m, 1 H), 6.98 (t, J = 7.9 Hz, 1 H), 6.50 (s, 1 H), 1.51
12.7 ppm; 19F NMR (470 MHz, CDCl3): δ = −74.7 (s, 3 F, (s, 9 H); 13C NMR (126 MHz, CDCl3): δ = 152.5, 139.7, 132.1,
CF3–TFA) ppm; HPLC (system A): tR = 5.1 min (purity 97% at 130.5, 127.3, 117.7, 94.4, 28.4, 27.5. All other spectroscopic
260 nm, 96% at 450 nm and 98% at 500 nm); LRMS (ESI+, data are identical to those reported by Viswanadham et al.39
recorded during RP-HPLC analysis): m/z 572.5 [M ]+° (100),
Unsymmetrical pyronin dye [2097130–10–2] (AR116).
mixture of 4-(diethylamino)salicylaldehyde (568 mg,
+
calcd for C38H42N3O2 572.3; LRMS (ESI−, recorded during
A
RP-HPLC analysis): m/z 616.3 [M + FA − 2H]− (100), calcd for 2.94 mmol, 1.7 equiv.), N-Boc-3-iodoaniline 11 (552 mg,
C39H42N3O4 616.3, and 662.4 [M + 2FA − 2H]− (40), calcd for 1.73 mmol,
1 equiv.), finely ground K3PO4 (732 mg,
−
−
C40H44N3O6 662.3; HRMS (ESI+): m/z 572.32597 [M]+°, calcd 3.45 mmol, 2 equiv.), CuI (33 mg, 0.17 mmol, 0.1 equiv.) and
+
for C38H42N3O2 572.32715; UV-vis: λmax (PB)/nm 548 (ε/dm3 picolinic acid (43 mg, 0.35 mmol, 0.2 equiv.) in dry DMSO
mol−1 cm−1 67 900); fluorescence λmax (PB)/nm 593 (ΦF ≪ 1%). (4.2 mL) was heated in a sealed tube at 90 °C overnight. The
Edaravone-based PGA-sensitive probe (10). Edaravone was reaction was checked for completion by TLC (eluent: DCM
used as a C-nucleophile (18.3 mg, 0.105 mmol, 1.05 equiv.). 100%, Rf = 0.50) and diluted with EtOAc. Then, the resulting
The reaction mixture was stirred at RT for 90 min, and then mixture was washed with deionized water thrice and brine,
under reflux for 2 h 30 min. The crude product was purified by dried over anhydrous Na2SO4, filtered and concentrated under
flash-column chromatography (step gradient of EtOAc in DCM reduced pressure. The resulting residue was purified by flash-
from 0% to 10%) and semi-preparative RP-HPLC (system G, tR column chromatography over silica gel (step gradient of EtOAc
= 41.0–43.0 min). The desired PGA-sensitive probe 10 was in heptane from 10% to 20%). The crude mixed bis-aryl ether
recovered (after freeze-drying) as a red amorphous powder 12 was directly dissolved in 55 : 45 (v/v) TFA–DCM (1.6 mL).
(10.3 mg, 18 μmol, yield 18%). IR (ATR): ν = 3298, 3064, 2974, The reaction mixture was stirred at RT for 30 min, and evapor-
2042, 2003, 1667, 1599, 1573, 1544, 1515, 1497, 1455, 1436, ated under reduced pressure. The resulting residue was puri-
1413, 1375, 1352, 1333, 1307, 1270, 1202, 1148, 1099, 1077, fied by flash-column chromatography (step gradient of MeOH
1024, 996, 963, 932, 863, 835, 766, 753, 721, 692, 670, in DCM from 0% to 10%) to provide pyronin AR116 as a dark
1
614 cm−1; H NMR (500 MHz, CDCl3): δ = 9.58 (d, J = 9.3 Hz, purple powder (65 mg, 0.24 mmol, overall yield for two steps
1 H), 7.94 (d, J = 8.0 Hz, 2 H), 7.73 (s, 1 H), 7.39 (d, J = 5.0 Hz, 14%). All spectroscopic data are identical to those recently
1 H), 7.37–7.30 (m, 4 H), 7.26 (t, J = 6.5 Hz, 3 H), 7.24–7.21 (m, reported by us.10
2 H), 7.20 (d, J = 1.3 Hz, 1 H), 7.09 (t, J = 7.6 Hz, 1 H), 6.72 (dt,
J = 7.3 Hz, J = 2.0 Hz, 1 H), 6.43 (dd, J = 9.4 Hz, J = 2.4 Hz, 1 H),
6.01 (d, J = 2.4 Hz, 1 H), 3.65 (s, 2 H), 3.27 (q, J = 7.2 Hz, 4 H),
Conflicts of interest
2.15 (s, 3 H), 1.07 (t, J = 7.1 Hz, 6 H) ppm; 13C NMR (126 MHz,
CDCl3): δ = 169.3, 163.1, 160.6, 157.7, 153.9, 151.8, 151.9, The authors declare no conflicts of interest.
140.2, 140.1, 139.5, 139.2, 136.8, 134.4, 130.2, 129.6, 129.3,
128.7, 127.8, 124.4, 120.3, 119.4, 115.1, 114.5, 113.31, 110.6,
107.6, 100.5, 45.1, 44.9, 13.5, 12.8 ppm; HPLC (system A): tR
6.1 min (purity 100% at 260 nm, 100% at 450 nm and 100% at
=
Acknowledgements
500 nm); LRMS (ESI+, recorded during RP-HPLC analysis): m/z This work is supported by the CNRS, Université de Bourgogne
559.3 [M + H]+ (100), calcd for C35H35N4O3 559.3; HRMS and Conseil Régional de Bourgogne through the “Plan
+
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d’Actions Régional pour l’innovation (PARI)” and the
“Fonds Européen de Développement Régional (FEDER)”
programs. S. D. gratefully acknowledges the Burgundy region
(“FABER” programme, PARI Action 6, SSTIC 6 “Imagerie,
instrumentation, chimie et applications biomédicales”) for his
Ph. D. grant (2014–2017). Financial support from Agence
Nationale de la Recherche (ANR, AAPG 2018, PRCI
LuminoManufacOligo, ANR-18-CE07–0045–01), especially for
the post-doc fellowship of K. R., the National Research
Foundation Singapore (NRF, LuminoManufacOligo, NRF2018-
NRF-ANR035) and GDR CNRS “Agents d’Imagerie Moléculaire”
(AIM) 2037 is also greatly acknowledged.
The authors thank the “Plateforme d’Analyse Chimique et
de Synthèse Moléculaire de l’Université de Bourgogne”
(PACSMUB, http://www.wpcm.fr) for access to spectroscopy
instrumentation. COBRA lab (UMR CNRS 6014) and Iris
Biotech company are warmly thanked for the generous gift of
some chemical reagents used in this work. The authors also
thank Marie-José Penouilh (University of Burgundy, PACSMUB)
for HRMS measurements, Dr. Myriam Laly (University of
Burgundy, PACSMUB) for the determination of TFA content in
samples purified by RP-HPLC, Dr. Ewen Bodio (University of
Burgundy, ICMUB, UMR CNRS 6302, OCS team) for access to
SAFAS Xenius XC spectrofluorimeter and Drs Valentin
Quesneau and Ibai E. Valverde (ICMUB, UMR CNRS 6302) for
helpful discussions and relevant comments on this manu-
script before publication.
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