Dual enzyme-responsive "turn-on" fluorescence sensing systems based on in situ formation of 7-hydroxy-2-iminocoumarin scaffolds

Article abstract of DOI:10.1039/c5ob01624j

A new strategy for the simultaneous fluorogenic detection of two distinct enzyme activities namely hydrolase (amidase or esterase) and reductase is described. This innovative biosensing method is based on the powerful "covalent-assembly" principle that in

Full text of DOI:10.1039/c5ob01624j

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Organic & Biomolecular Chemistry
PAPER
Dual enzyme-responsive "turn-on" fluorescence sensing systems
based on in situ formation of 7-hydroxy-2-iminocoumarin
scaffolds†
Received 00th January 20xx,
Accepted 00th January 20xx
Sylvain Debieu,a and Anthony Romieu*a,b
DOI: 10.1039/x0xx00000x
A new strategy for the simultaneous fluorogenic detection of two distinct enzyme activities namely hydrolase (amidase or
esterase) and reductase is described. This innovative biosensing method is based on the powerful "covalent-assembly"
principle that involves in situ synthesis of a fluorophore from a non-fluorescent caged precursor and through domino
reactions triggered by the two analytes of interest. To establish this approach, penicillin G acylase (PGA) (or pig liver
esterase (PLE)) and nitroreductase (NTR) were chosen as model enzymes, and original bis-O-protected 2,4-
dihydroxycinnamonitrile derivatives acting as dual-reactive probes readily convertible to highly fluorescent 7-hydroxy-2-
iminocoumarin scaffolds upon reacting with the two selected enzymes, were synthesised. The two phenolic groups
available within the core structure of these probes play a pivotal role in generating iminocoumarin scaffold through an
intramolecular cyclisation reaction (hydroxyl group in C-2 position) and in enhancing its push-pull character (hydroxyl
group in C-4 position). Their orthogonal and temporary protection with two different enzyme-labile masking groups is the
cornerstone in the design of this novel class of fluorogenic "turn-on" probes. Their evaluation using fluorescence-based in
vitro assays and HPLC-fluorescence/-MS analyses have enabled us both to demonstrate the claimed activation mechanism
(in particular the specific order in which the two enzymes react with the probe) and to highlight the potential utility of
www.rsc.org/
these
advanced
chemical
tools
in
multi-analyte
sensing
applications.
incorporation of multiple reacting/binding sites within a
unique photoactive molecular scaffold. That is able to
generate different fluorescence output signals (distinct
spectral responses via the same or different wavelength
channels) upon its reaction with several distinct analytes.
Molecular probes with a single reacting/binding site, capable
of producing distinct optical outputs depending on the analyte
being reacted, are also considered. Thus, numerous dual
fluorogenic probes for sensing two species from among some
metal cations, anions and/or neutral biorelevant molecules
(e.g., biothiols), through specific chemical reactions occurring
Introduction
Fluorescent probes for dual- or multi-analyte detection have
recently emerged as valuable and powerful analytical tools for
(bio)sensing and bioimaging applications. Indeed, they have
clear advantages over single-analyte responsive probes used
either sequentially or simultaneously, particularly to improve
the power of analytic assays (in terms of throughput,
spatial/temporal resolution and quantitative response) but
also facilitate their implementation in molecular-scale devices,
through atom economy and high-density integration. Among
1
2
either simultaneously or consecutively, have been reported
the host of useful and promising applications recently claimed
for such fluorescent molecular logic gates, "smart" medical
1
during the last decade. By contrast, little attention has been
paid to reaction-based fluorescent probes enabling two
different enzyme activities monitoring within the same sample
or biological medium, whereas they are valuable tools to
improve the diagnosis accuracy (avoiding false positives) and
expedite the right treatment of diseases. Particularly
noteworthy are two examples of FRET-based probe for
simultaneous (or sequential) intracellular detection of β-D-
glucosidase and phosphodiesterase I, and two distinct
diagnostics relying on the simultaneous quantitative detection
of several analytes to one pathology is often highlighted. The
design principle commonly adopted for constructing such
fluorescent chemodosimeters/chemosensors is based on the
3
proteases namely matrix metalloproteinase 2 (MMP-2) and
4
caspase-3, respectively. This probe principle may find further
applications in multi-enzyme analysis by changing the sensitive
linkers and/or fluorophore pairs, but will remain limited to
hydrolases detection. More recently, a brilliant concept for in
vivo bioluminescence detection of H2O2 and caspase-8 (an
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Journal Name
19
apoptosis-related cysteine protease), two major contributors amino/7-hydroxy-(2-imino)coumarins family.
Indeed, non-
View Article Online
to oxidative stress and cell death in injury and disease, has fluorescent precursors readily coDnOveI:r1t0ib.1l0e39/Ci5nOtoB0162(42J-
a specific cyclisation reaction
formation of D-luciferin from two caged precursors, each triggered by a reactive analyte are already well known.
5
been reported by Bertozzi, Chang and co-workers. In situ imino)coumarins through
14b,
14c, 14e-g, 14i-k, 14m, 14o-r
specifically activated by one of the two targeted analytes, and
Furthermore, since 7-amino-/7-
6
through the CBT-based "click" reaction , is the cornerstone of hydroxy-(2-imino)coumarins are known to be fluorogenic dyes
this dual-analyte imaging strategy. Interestingly, the recent (7-NH2/7-OH moiety acting as a fluorescence "OFF-ON" switch
availability of enzyme-responsive caged luciferins (i.e., luciferin based on its protection-deprotection), one would assume that
derivatives whose the 6-NH2 or 6-OH is masked by an enzyme- further functionalization of the (2-imino)coumarin precursor
7 8, 9, 10
,
labile moiety)
should facilitate the further extension with an aniline or a phenol moiety masked by a trigger
of this "self-assembly" bioluminogenic approach to recognition unit reactive toward a second distinct analyte
simultaneous imaging of two relevant enzymes in living could easily lead to reaction-based probes acting as "AND"
3 20
,
systems.
fluorogenic logic gates (Figure 1).
In this context, we are interested in devising a new fluorogenic In this paper, we report the practical implementation of this
platform engineered to respond only in the presence of two innovative dual-sensing strategy using hydrolase (PGA or pig
distinct enzymes and to generate an intense "turn-on" liver esterase (PLE)) and nitroreductase (NTR) as enzyme pair
fluorescence signal as a result of domino reactions triggered by model. This had led to the preparation of three dual-reactive
these two analytes. Indeed, it is now well established that probes whose the activation mechanism was studied in detail
reaction-based small-molecule fluorescent probes (also known through in vitro fluorescence assays and HPLC-fluorescence/-
as latent fluorophores, pro-fluorophores or fluorescent MS analyses, particularly to demonstrate in situ formation of
chemodosimeters) are powerful tools for detecting/imaging a blue-emitting 2-iminocoumarin scaffolds mediated by
myriad of analytes including enzymes, biomolecules, chemical combination of the two enzymes.
11, 12
pollutants, cations and anions.
Among the various
organic reaction-based strategies currently implemented in
Results and discussion
the field of molecular (bio)sensing, the "covalent assembly"
13
approach
using the analyte of interest to carry out the
General considerations for the design of dual-enzyme
fluorogenic "turn-on" probes - Selection of structural units
As suggested above, we have decided to focus on the design of
molecular probes capable of generating a fluorescent dye
belonging to the family of 3-substituted-7-hydroxy-2-
iminocoumarins, upon dual-enzymatic activation. Indeed, 7-
hydroxycoumarin derivatives are often more fluorescent than
their 7-amino counterparts, particularly at physiological pH,
and the facile functionalization of their C-3 position with an
electron-withdrawing group (typically, a cyano group) or an
heteroaryl substituent (e.g., 2-benzothiazolyl or 2-
benzimidazolyl moiety) is an asset to red-shift their
fluorophore synthesis in situ from non-fluorescent caged
precursor(s), is a very promising way to readily obtain highly
fluorimetric "turn-on" signal from zero background. Thus, this
concept was successfully applied for the detection of various
analytes including enzymes (monoamine oxidases (MAO A and
B), penicillin G acylase (PGA), phosphatases), cations/anions
-
(Cu(II), Hg(II), MeHg(I), F- and NO2 ), sarin mimics, H2S, NO and
peroxynitrite, through the formation of pyrrolo-coumarin,
pyrazino-benz[e]indole,
7-N,N-dialkylaminocoumarin,
2-
arylbenzotriazole, pyronin B, benzo[c]cinnoline and resorufin
13, 14 15
,
fluorescent scaffolds.
Further implementation of this
strategy to fluorogenic bimolecular reactions producing red-
emitting cyanine dyes, was recently reported for illuminating
absorption/emission maxima, and to facilitate fluorescence
21, 22
detection in complex biological media.
Furthermore, bis-
16
17
G-quadruplex DNA structures and protein CRABPII. It is
also worth mentioning that the more traditional strategy
O-protected 2,4-dihydroxycinnamonitrile derivatives should be
easily synthesised from cheap commercial starting materials (i.
based on protection-deprotection of the key functional group
e.,
2,4-dihydroxybenzaldehyde
and
malonitrile
or
12
of a fluorophore (typically, a phenol or an aniline moiety)
benzothiazole-2-acetonitrile) and through a Knoevenagel-type
was recently used by Prost and Hasserodt to develop the
condensation reaction. Consequently, the two fluorophores
18
23
appealing "double gating" concept.
The activation by
namely
3-(2-benzothiazolyl)-7-hydroxy-2-iminocoumarin
24, 25
successive cleavage of a pre-pro-fluorophore with two distinct
enzymes namely β-D-galactosidase and leucine
aminopeptidase leads to the release of a phenol-based
fluorophore.
From our side, we wished to apply the "covalent assembly"
probe design principle to reach the same purpose. To this end,
a fluorophore precursor bearing two distinct reactive sites, one
involved in the formation of the conjugated backbone and one
for enhancing the push-pull character, masked by two
different enzyme-labile protecting groups, is needed (Figure 1).
The most convenient class of fluorophores to be used to
rapidly provide a proof-of-concept is undoubtedly the 7-
and 3-cyano-7-hydroxy-2-iminocoumarin
have been
selected as fluorescent reporters for this study (Figure 1). For
in vitro validations performed under simulated physiological
conditions (i.e., PB, pH 7.5), the significant hydrophobic
character of the claimed coumarin precursor is a potential
issue. So, we had initially planned to design fluorogenic "turn-
on" probes reactive toward both NTR and a sulfatase. Indeed,
this latter class of hydrolytic enzymes is known to catalyse the
hydrolysis of sulfate esters whose the inherent hydrophilicity is
26
beneficial for the overall aqueous solubility of the probes.
Unfortunately, our preliminary works have revealed that dual
NTR-sulfatase fluorogenic "turn-on" probes are completely
2 | Org. Biomol. Chem., 2015, 00, 1-3
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unstable in aqueous solution and a rapid hydrolysis of their respectively). Pre-activation of Ac-PHBA was achieved by
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DOI: 10.1039/C5OB01624J
aryl sulfate ester moiety occurred. Alternatively, we have treatment with diphosgene in dry DCM in presence of TEA. The
chosen to work with (i) PGA (also known as penicillin amidase) resulting crude chloroformate was reacted with 3 to provide
a commercially available and widely used biocatalyst in the the targeted dual PLE-NTR fluorogenic "turn-on" probe 5 in
enzymatic synthesis of β-lactam antibiotics, since it allows for 32% yield. Interestingly, this benzothiazolyl-based probe 5 was
27
the deprotection of phenylacetyl-protected amines , and (ii) obtained as a mixture of E/Z isomers (56 : 44 and 85 : 15
pig liver esterase (PLE), known to catalyse the hydrolysis of determined by HPLC and NMR respectively ; difference in this
acetate esters and commonly used for kinetics resolution and ratio value was explained by the lower solubility of less polar
28
asymmetric synthesis in organic chemistry , in place of isomer in RP-HPLC eluents and different UV-vis absorption
sulfatases. However, the use of these two model enzymes properties between two isomers (both wavelength maximum
directly impacts the structural requirements of reaction-based and molar extinction coefficient)). This synthetic methodology
probes. Indeed, to transfer the amide bond-breaking event to was also applied to PhAc-PABA but failed (due to the
a phenol release, the use of a self-immolative spacer is premature degradation of PhAc-PABA throughout its activation
29
required.
para-Aminobenzyl alcohol (PABA) was chosen with diphosgene). No further attempts were made to obtain
because it has been successfully used to conjugate peptide PGA-NTR benzothiazolyl-based probe because we suspected
substrates to masked fluorophores, to obtain valuable that its high hydrophobic character would be prohibitive for
fluorogenic reagents for protease assays and/or in vivo applications in aqueous buffers. For the dicyano probe 8 and 9,
30
molecular
imaging
applications.
Thus,
para- diphosgene was first reacted with phenol 4 and benzyl alcohol
(phenylacetamido)benzyl moiety was selected as the PGA- Ac-PHBA/PhAc-PABA was next introduced (particularly to
labile phenol protecting group. To enhance the overall stability avoid degradation of PhAc-PABA) but the desired products
of the phenol-based probes bearing an esterase-sensitive were recovered in poor yields (11% and 9% respectively).
moiety, it is also recommended to introduce a self-immolative These results were unsatisfactory, so pre-activation of Ac-
spacer (typically, para-hydroxybenzyl alcohol (PHBA)) between PHBA/PhAc-PABA with para-nitrophenyl chloroformate in dry
the acetyl and phenol moieties. Consequently, para- THF in the presence of TEA was investigated. The resulting
34
35
(acetoxy)benzyl (Ac-PHBA) was selected as the PLE-labile para-nitrophenyl carbonate derivatives 6
phenol protecting group.
and 7
were
8, 31
In both cases, the bio-labile purified and reacted with 4 to provide the two targeted dual
moiety will be introduced onto the phenol via a carbonate fluorogenic "turn-on" probes 8 and 9 with improved 30% and
linkage because preliminary etherification-based synthetic 17% yields respectively. All spectroscopic data (see
strategies failed to give the desired protected phenol Experimental section and ESI†), especially IR, NMR and mass
derivatives (vide infra). Finally, we have regarded the masking spectrometry, were in agreement with the structures assigned.
of the second phenol function by the para-nitrobenzyl moiety, Their purity was checked by RP-HPLC and found to be in the
which is a commonly used caging group that targets reductase range 94-96%. It was not feasible to perform further RP-HPLC
10, 32
activity for sensor and prodrug development.
At this purification (typically, the method best suited to increase this
stage, it is also important to point out the preferred phenolic percentage purity to 100%) due to hydrophobicity and
position for introducing each enzyme-labile trigger-spacer moderate stability of 5, 8 and 9 in aqueous acidic mobile
combined unit: 2-OH for para-nitrobenzyl and 4-OH for para- phases.
(phenylacetamido)benzyl and para-(acetoxy)benzyl, arising
from our preliminary synthetic attempts and shortcomings
encountered (Figure 1).
Photophysical characterization and dual-enzymatic activation
of fluorogenic "turn-on" probes - Fluorescence-based assays
UV-vis absorption spectra of compounds 5, 8 and 9 were
Synthesis of bis-O-protected 2,4-dihydroxycinnamonitrile
derivatives
recorded in phosphate buffer (PB, 0.1 M, pH 7.5, simulated
36
physiological conditions). They exhibited a broad absorption
The three dual-enzyme fluorogenic "turn-on" probes were
spectrum covering virtually the entire UV spectral range (250-
synthesised in four steps (Scheme 1). First, the 4-OH group of
450 nm), and characterized by a maximum centered at 372-
2,4-dihydroxybenzaldehyde was selectively protected as silyl
383 nm depending on the nature of C-3 substituent (see Figure
ether using standard conditions. The resulting TBDMS ether 1
S1†). This spectral pattern is in part due to the formation of
was next reacted with an excess of para-nitrobenzyl bromide
aggregates promoted by the hydrophobic character of these
(5 equiv.) under mild "Kuhn methylation" conditions (i.e., the
cinnamonitrile derivatives. This aggregation behaviour
33
use of Ag2O as electrophilic catalyst and base) , to readily
prevented us from obtaining linear relationships between
introduce the NTR-labile protecting group into the 2-OH
absorbance and concentration required to determine molar
position, without premature deprotection of the 4-OH group.
extinction coefficients of these absorbing species. As expected,
The full-protected benzaldehyde derivative 2 was condensed
these three probes, without exception, were not fluorescent
with either benzothiazole-2-acetonitrile or malonitrile in EtOH
under simulated physiological conditions and their emission
and in the presence of a catalytic amount of piperidine, to give
spectrum exhibits a zero-baseline remaining constant over
the Knoevenagel adducts which were desilylated by treatment
time (see Figures S4-S6†).
with TBAF in THF. The corresponding phenols 3 and 4 were
obtained in satisfying yields (38% and 55% for the two steps,
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Concerning the dual-enzymatic activation of 5, 8 and 9, fluorescence activation monitoring the formVaietwioAnrticleoOf nli7ne-
fluorescence-based in vitro assays were performed with hydroxy-2-iminocoumarin scaffold was oDbOseI:r1v0e.1d03w9h/Ce5nO8B0a1n62d49J
commercial enzymes (recombinant NTR from E. coli used with were incubated with both enzymes simultaneously or better
co-factor NADH, recombinant PGA from Alcaligenes faecalis according the sequential mode: pre-incubation with hydrolase
and esterase from porcine liver). We were mindful that (10 min) followed by addition of NTR/NADH. Only the
37
fluorescence of NADH (Abs./Em. maxima = 340/470 nm)
sequential assay involving pre-incubation of the probes with
may cause interference with enzyme-induced fluorogenic NTR led to poor fluorescence activation. Interestingly, further
signal but the excitation channel selected for the probes (455 addition of compound 4 (a good fluorogenic substrate of NTR)
nm for 5 and 418 nm for 8 and 9) is far enough away from into this latter enzymatic reaction mixture did not led to a
absorption maximum of co-factor to prevent its photonic dramatic change in fluorescence signal (see Figure S7†). These
excitation and subsequent emission. For each pro-fluorophore, results supports the hypothesis that compounds 5 and 8 are
different scenarios were tested under simulated physiological effective at blocking the activity of this reductase. However, a
conditions (PB, pH 7.5, 37 °C). Concerning benzothiazolyl- deeper understanding of this inhibition mechanism requires
based probe 5, fluorescence emission time-course further enzyme kinetics and molecular docking experiments
measurements were achieved with the following enzyme that will soon be carried out and reported in due course.
addition procedures: (i) NTR/NADH (20 min) then PLE, (ii) PLE To confirm that the resulting fluorescence emission was due to
(10 min) then NTR/NADH, (iii) NTR/NADH and PLE 7-hydroxy-2-iminocoumarin dye formed in situ, all enzymatic
simultaneously, and without enzyme (Figure 2A). We also reaction mixtures were directly analysed by RP-HPLC coupled
checked that co-factor alone did not trigger phenol with fluorescence detection using aqueous triethylammonium
deprotection resulting in fluorogenic response, by carrying out bicarbonate buffer (TEAB, 50 mM, pH 7.5) and CH3CN as
some assays that involved pre-incubation of probe with NADH eluents (see Figures 3 and S9† for illustrative examples of RP-
before adding enzymes. A rapid and gradual increase of blue- HPLC elution profiles and Figure S8† for negative controls with
green fluorescence intensity at 489 nm (Ex. 455 nm) fully starting non-fluorescent probes). The use of a neutral aqueous
consistent with in situ formation of 3-(2-benzothiazolyl)-7- mobile phase is required to ensure phenol deprotonation of
hydroxy-2-iminocoumarin was observed when 5 is pre- newly formed 7-hydroxy-2-iminocoumarins and maximize their
incubated with PLE before adding NADH and NTR. It was fluorescence properties and thus their detection. Furthermore,
somewhat disappointing to note that probe's activation was the facile synthesis of authentic samples of 3-(2-
23
much less effective with two other incubation protocols. These benzothiazolyl)-7-hydroxy-2-iminocoumarin
,
3-(2-
23
results
suggested
that
this
full-protected
2,4- benzothiazolyl)-7-hydroxycoumarin
,
3-cyano-7-hydroxy-2-
24, 25
dihydroxycinnamonitrile derivative may be a poor substrate of iminocoumarin and 3-cyano-7-hydroxycoumarin
, has
NTR or act as an effective competitive inhibitor of this enzyme, enabled us to have reference compounds for such liquid
and is reactive toward this reductase only once its 4-OH chromatographic analyses. Thus, the identification of peaks
phenol group is free. Curiously,
fluorescence was observed when the probe is pre-incubated standards. In the case of benzothiazolyl-based probe 5, the
only with NTR/NADH. The presence of a minute amount of formation of both 3-(2-benzothiazolyl)-7-hydroxy-2-
a slight increase in was done by comparison of retention times with those of
free 4-OH 2,4-dihydroxycinnamonitrile derivative 3 in the iminocoumarin and 3-(2-benzothiazolyl)-7-hydroxycoumarin
sample (reminder: purity 96%) or its minor formation in PB at was confirmed (Table 1, entries 1 and 2). The side-formation of
pH 7.5 was suspected to explain this premature fluorescent coumarin through the non-enzymatic hydrolysis of the imine
activation by
a single enzyme. However, the lack of moiety occurred during the enzymatic reaction and HPLC
fluorescence increase observed for the control reaction analysis. This is not a problem for the claimed dual-enzyme
involving incubation of 5 with buffer alone, illustrates one of sensing scheme because this also a strongly emissive species
the major benefits of the dual-reactive fluorogenic probes (Table 1, entries 1 and 2). For bis-cyano-based probes 8 and 9,
compared to pro-fluorophores activated through a single unambiguous
identification
of
3-cyano-7-hydroxy-(2-
(bio)chemical event. Even though the overall stability of such imino)coumarins was also done but a further minor broad split
fluorogenic platforms may not necessarily be remarkable peak was observed on the RP-HPLC elution profiles, which may
under physiological conditions, the probability of obtaining assigned to 3-carbamoyl-7-hydroxy-(2-imino)coumarins (Figure
false positive fluorescence signals is lower because this would S9† and Table 1, entries 3-5). Indeed, the nitrile moiety
require the non-specific hydrolysis of their two enzyme- introduced onto the C-3 position of coumarins, is prone to
24
responsive caging/quenching groups. Better results were facile hydrolysis.
The non-enzymatic conversion of 2-
obtained with cyano-based probes 8 and 9. Time-dependant iminocoumarin derivative into the corresponding coumarin is
fluorescence analyses were performed under the same particularly beneficial because the relative fluorescence
conditions than those described above, except for the quantum yield of this latter product was found to be
excitation/emission wavelength channel (Ex./Em. 418/458 significantly higher than that of its parent imine (Table 1,
nm), which was tuned for an optimal detection of 3-cyano-7- entries 3-5).
hydroxy-2-iminocoumarin (Figure 2B-C). Furthermore, for the
4-O-(para-(phenylacetamido)benzyloxycarbonyl) derivative 9,
PGA was used instead of PLE. In both cases, a strong blue
Dual-enzymatic activation of fluorogenic "turn-on" probes -
Mechanism study through RP-HPLC-MS analyses
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To confirm the sensing process claimed in Figure 1, HPLC-MS For the first time, we have demonstrated that the "covalent-
View Article Online
DOI: 10.1039/C5OB01624J
analyses of the enzymatic hydrolysis mixtures were performed. assembly" probe design principle could be successfully applied
To carry out mass detection through the "full-scan" detection for the simultaneous fluorogenic detection of two different
mode (more practical and convenient but less sensitive than enzyme activities namely hydrolase and reductase. The double
the single-ion monitoring (SIM) mode), enzyme reactions were activation of these reactive probes through hydrolysis of
performed at a higher concentration than this previously used amide (or ester) bond and nitro-reduction, followed by domino
for fluorescence-based assays (85 µM against 1.0 µM). It was 1,6-elimination/cyclisation reactions, was efficient at
not possible to get reproducible results with the generating strongly fluorescent 7-hydroxy-(2-imino)coumarins.
benzothiazolyl-based probe 5 due to its poor solubility in PB at These sophisticated biosensing systems act as AND-type
this concentration. Consequently, such mechanistic study was molecular logic gates because fluorescence signal production
focused only on less hydrophobic bis-cyano-based probes 8 occurred only in the presence of both analytes. To rapidly
and 9 (Figures 4 and S10-S12†). The first valuable information apply this promising concept to molecular imaging of disease-
disclosed by these HPLC-MS analyses concerned the stability of biomarkers pairs for diagnostic purposes, extension of the
the probes in the reaction buffer. Indeed, as shown in Figures "doubly-triggered covalent assembly" approach to longer
4B and S10†, following incubation at pH 7.5 without enzymes, wavelength fluorophores (e. g., pyronin B or far-red cyanine
39
probes 8 and 9 were partly degraded to a single product dyes ) is currently in progress in our lab. Furthermore, the
identified as the corresponding benzaldehyde derivative (Table dual-reactive 7-hydroxy-(2-imino)coumarin precursors of this
1, entries 6 and 7). Under these mild conditions, retro- work can be easily modified to rapidly obtain "dual-lock"
Knoevenagel reaction is able to occur but curiously, such side- structures combining two distinct reactive/quenching groups
reaction was never reported in publications describing O- sensitive to the same analyte. Indeed, this is the preferred
protected 2-hydroxycinnamonitrile derivatives as effective approach for designing reaction-based fluorescent probes with
38
reaction-based fluorescent probes. This does not challenge unprecedented performances, especially those related to the
detection mechanisms based on in situ formation of 2- effective discrimination between molecules exhibiting similar
40 41
,
iminocoumarins because the formed benzaldehyde-type chemical reactivity (e.g., biothiols).
derivative remains non-fluorescent, but it is important to bear
in mind that this may prevent quantitative determination of
targeted analyte through the decrease of the "active" probe
Experimental section
concentration. In the future, this should be addressed in
studies focused on the use of benzylidenemalonitrile-based
probes as chemodosimeters. Gratifyingly, no trace of free 2-
OH and/or 4-OH 2,4-dihydroxycinnamonitrile derivatives that
might lead to 7-hydroxy-(2-imino)coumarins without
enzymatic action were detected in these blank samples. This
was a bit in contrast with conclusions drawn from fluorescent
measurements related to enzyme assays involving pre-
incubation with NTR/NADH followed by addition of hydrolase
(vide supra), but explained by the differences in probe's
concentration used in both assays (85 µM against 1.0 µM). The
preferential reaction of probes with hydrolase against NTR was
clearly demonstrated through the detection of 2,4-
dihydroxycinnamonitrile derivative having free 4-OH and 2-OH
always masked as para-nitrobenzyl ether (Figure 4C and Table
1, entry 8). A similar trend was observed with the retro-
Knoevenagel products as evidenced by the detection of 2-O-
(para-nitrobenzyl)-2,4-dihydroxybenzladehyde (Figure 4D and
Table 1, entry 9). The intermediate was next deprotected onto
their 2-OH position under the action of NTR/NADH, to undergo
rapid spontaneous cyclisation leading to 7-hydroxy-(2-
imino)coumarins bearing either the cyano or carbamoyl group
as C-3 substituent (Figure 4D and table 1, entries 3-5). We
were therefore able to demonstrate that in situ synthesis of
the fluorophore scaffold is really triggered by two distinct
enzyme activities through a sequential mode of action.
General
Unless otherwise noted, all commercially available reagents
and solvents were used without further purification. TLC were
carried out on Merck DC Kieselgel 60 F-254 aluminum sheets.
The spots were directly visualised or through illumination with
UV lamp (λ
= 254/365 nm) and/or staining with a
phosphomolybdic acid solution (4.8% wt. in EtOH). Column
chromatography purifications were performed on silica gel
(63-200 µm) from Sigma-Aldrich (technical grade). All solvents
were dried by standard procedures [DCM: distillation from
CaH2; absolute EtOH: storage over anhydrous Na2SO4; THF:
distillation from sodium benzophenone diketyl; and TEA:
storage over anhydrous Na2SO4]. Anhydrous DMF was
purchased from Carlo Erba, and stored over 4 Å molecular
sieves. Peptide synthesis grade DIEA was provided by Iris
Biotech GmbH. The HPLC-gradient grade acetonitrile (CH3CN)
was obtained from Carlo Erba. Formic acid (FA, grade "eluent
additive for LC-MS") was provided by Sigma-Aldrich.
Phosphate buffer (100 mM, pH 7.5) and aq. mobile-phases for
HPLC were prepared using water purified with a PURELAB
Ultra system from ELGA (purified to 18.2 MΩ.cm). PLE
(esterase from porcine liver, E3019, 27 U/mg of lyophilized
enzyme), NTR (from E. coli, N9284, 0.1 U/μg, lyophilized
enzyme + buffer resuspended in ultrapure water) and NADH
were purchased from Sigma-Aldrich and stored at -20 °C.
Recombinant PGA (from Alcaligenes faecalis) was provided by
Universität Hohenheim (0.63 U/mg of lyophilized enzyme).
Triethylammonium bicarbonate (TEAB, 1.0 M) buffer was
prepared from distilled TEA and CO2 gas. 3-(2-Benzothiazolyl)-
Conclusions
This journal is © The Royal Society of Chemistry 20xx
Org. Biomol. Chem., 2015, 00, 1-3 | 5
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ARTICLE
Journal Name
7-hydroxycoumarin,
iminocoumarin,
3-(2-benzothiazolyl)-7-hydroxy-2- Several chromatographic systems were used for the analytical
View Article Online
DOI: 10.1039/C5OB01624J
3-cyano-7-hydroxycoumarin,
3-cyano-7- experiments (HPLC-MS or HPLC-fluorescence): System A: RP-
hydroxy-2-iminocoumarin were prepared according to HPLC-MS (Phenomenex Kinetex C18 column, 2.6 µm, 2.1 × 50
23-25
literature procedures.
PHBA and PhAc-PABA.
See ESI† for the synthesis of Ac- mm) with CH3CN (+ 0.1% FA) and 0.1% aq. FA (pH 3.2) as
eluents [linear gradient from 5% to 100% (5 min) of CH3CN
followed by isochratic at 100% (1.5 min)] at a flow rate of 0.5
mL min-1. UV-visible detection was achieved at 220, 260, 300
and 360 nm (+ diode array detection in the range 220-500 nm).
Instrument and methods
Centrifugation steps were performed with an Hettich Universal
ESI-MS detection in the positive/negative mode ("full scan",
150-1500 a.m.u., data type: centroid, needle voltage: 3.0 kV,
detector voltage: 1100 V, probe temperature: 350 °C, cone
voltage: 75 V and scan time: 1 s). System B: System A with 100-
700 a.m.u for "full scan" mass detection. System C: System A
with the following gradient [0% CH3CN (2 min) followed by
linear gradient from 0% to 100% (6 min) of CH3CN followed by
isochratic at 100% (1 min)]. UV-visible detection was achieved
at 220, 260, 350 and 418 nm (+ diode array detection in the
range 220-500 nm). System D: RP-HPLC-fluorescence
(Phenomenex Kinetex C18 column, 2.6 µm, 2.1 × 50 mm) with
CH3CN and aq. TEAB (50 mM, pH 7.5) as eluents [0% CH3CN (1
min) followed by linear gradient from 0% to 100% (5 min) of
CH3CN followed by isochratic at 100%] at a flow rate of 0.5 mL
min-1. Fluorescence detection was achieved at 45 °C at the
following Ex. /Em. channels: 350/460 nm and 418/458 nm
(sensitivity: 1, PMT 1, filter wheel: auto). System E: System D
with the following Ex./Em. channels for fluorescence
detection: 350/460 nm, 431/488 nm and 455/489 nm.
1
320 instrument. H- and 13C-NMR spectra of compounds 1-9
were recorded either on a Bruker Avance 300 or on a Bruker
Avance 500 spectrometer. Chemical shifts are expressed in
parts per million (ppm) from the residual non-deuterated
42
solvent signal. J values are expressed in Hz. IR spectra were
recorded with a Bruker Alpha FT-IR spectrometer equipped
with an universal ATR sampling accessory. The bond
vibration frequencies are expressed in reciprocal centimeters
(cm-1). HPLC-MS analyses were performed on a Thermo-Dionex
Ultimate 3000 instrument (pump + autosampler) equipped
with a diode array detector (Thermo-Dionex DAD 3000-RS)
and a MSQ Plus single quadrupole mass spectrometer (LRMS
analyses through ESI). HPLC-fluorescence analyses were
performed with the same instrument coupled to a RS
fluorescence detector (Thermo-Dionex, FLD 3400-RS). High-
resolution mass spectra (HRMS) were recorded on a Thermo
LTQ Orbitrap XL apparatus equipped with an electrospray
ionisation (ESI) source. UV-visible spectra were obtained either
on a Varian Cary 50 scan spectrophotometer by using a
rectangular quartz cell (Hellma, 100-QS, 45 × 12.5 × 12.5 mm,
pathlength 10 mm, chamber volume: 3.5 mL). Fluorescence
spectroscopic studies (emission/excitation spectra and
kinetics) were performed with an HORIBA Jobin Yvon Fluorolog
spectrophotometer (software FluoEssence) with a standard
fluorometer cell (Labbox, LB Q, 10 mm). Emission spectra were
recorded under the same conditions after excitation at the
corresponding wavelength (shutter: Auto Open, excitation slit
= 5 nm and emission slit = 5 nm). All fluorescence spectra were
corrected.
4-((tert-Butyldimethylsilyl)oxy)-2-hydroxybenzaldehyde
44
(1). Under Ar atmosphere, to a stirred solution of TBDMS-Cl
(2.20 g, 14.4 mmol, 1 equiv.) in dry DCM (40 mL) was added
imidazole (1.15 g, 15.8 mmol, 1.1 equiv.). After 5 min of
stirring, 2,4-dihydroxybenzaldehyde (2 g, 14.4 mmol, 1 equiv.)
was added and the resulting reaction mixture was stirred at RT
for 4 h. Thereafter, the reaction mixture was washed with
deionised water and brine, dried over anhydrous MgSO4, and
concentrated under vacuum. The resulting residue was
purified by chromatography on a silica gel column (PE-EtOAc,
step gradient from 100 to 95 : 5, v/v) to give the desired silyl
ether 1 as colourless oil (2.62 g, yield 72%). Rf 0.61 (PE-EtOAc,
98 : 2, v/v); δH(300 MHz, CDCl3) 11.33 (s, 1 H), 9.72 (s, 1 H),
7.40 (d, J 8.7, 1 H), 6.46 (dd, J 8.7, J 2.4, 1 H), 6.38 (d, J 2.4, 1
H), 0.98 (s, 9 H), 0.25 (s, 6 H).
Fluorescence quantum yields of 3-cyano-7-hydroxycoumarin
and 3-cyano-7-hydroxy-2-iminocoumarin were measured at 25
°C by a relative method using quinine sulfate (ΦF = 60% in 0.05
M H2SO4) as a standard (for the corresponding Abs/Ex/Em
spectra, see Figures S2-S3†). The following equation was
43
used to determine the relative fluorescence quantum yield:
4-((tert-Butyldimethylsilyl)oxy)-2-((4-nitrobenzyl)oxy)-
benzaldehyde (2). To a stirred solution of phenol 1 (1 g, 3.96
mmol, 1 equiv.) in Et2O (17 mL) were sequentially added 4-
nitrobenzyl bromide (4.28 g, 19.8 mmol, 5 equiv.) and Ag2O
(3.6 g, 15.8 mmol, 4 equiv.) at RT. The resulting reaction
mixture was stirred overnight. Thereafter, the crude was
directly purified by chromatography on a silica gel column
(eluent: PE-EtOAc, step gradient from 100 to 90.7 : 0.3, v/v to
completely remove the large excess of 4-nitrobenzyl bromide
and after a step gradient from 95 : 5 to 85 : 15, v/v) to afford
the desired product 2 as white solid (1.35 g, yield 88%). Rf 0.78
(DCM); νmax(neat)/cm-1 2952, 2927, 2855, 1683, 1593, 1340,
1256; δH(300 MHz, CDCl3) 10.38 (s, 1 H), 8.28 (d, J 9.0, 2 H),
ΦF(x) = (AS/AX)(FX/FS)(nX/nS)2ΦF(s)
where A is the absorbance (in the range of 0.01-0.1 A.U.), F is
the area under the emission curve, n is the refractive index of
the solvents (at 25 °C) used in measurements, and the
s and x represent standard and unknown,
respectively. The following refractive index value is used: 1.333
for aq. H2SO4 and 1.337 for PB.
subscripts
High-performance liquid chromatography separations
6 | Org. Biomol. Chem., 2015, 00, 1-3
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Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
7.79 (d, J 8.7, 1 H), 7.63 (d, J 8.7, 2 H), 6.53 (dd, J 9.3, J 2.1, 1 filtration to obtain the desired product 4 as orange solid (460
View Article Online
-1
DOI: 10.1039/C5OB01624J
H), 6.39 (d, J 2.4, 1 H), 5.24 (s, 2 H), 0.96 (s, 9 H), 0.21 (s, 6 H); mg, yield 55%). Rf 0.5 (DCM-EtOAc, 9 : 1, v/v); νmax(neat)/cm
δC(75 MHz, CDCl3) 187.9, 163.0, 161.8, 147.7, 143.4, 131.0, 3335, 2221, 1605, 1561, 1513, 1354, 1329, 1289; δH(300 MHz,
127.5, 124.0, 119.6, 113.5, 104.5, 69.0, 25.5, 18.3, -4.3; HPLC DMSO-d6) 11.10 (bs, 1 H, OH), 8.31 (s, 1 H), 8.28 (d, J 8.7, 2 H),
(system A): tR = 6.2 min, purity = 100% (at 260 nm); λmax 8.05 (d, J 9.0, 1 H), 7.77 (d, J 8.7, 2 H), 6.62 (dd, J 8.7, J 2.1, 1
(recorded during the HPLC analysis)/nm 227, 272 and 306; H), 6.57 (d, J 2.1, 1 H), 5.40 (s, 2 H); δC(125 MHz, DMSO-d6)
LRMS (ESI+): m/z 388.3 [M + H]+ (100) and 641.1 [M + H + 166.6, 160.6, 153.9, 147.7, 144.27, 131.0, 120.9, 124.1, 116.0,
CH3CN]+ (80), calcd for C20H25NO5Si 387.1.
115.0, 112.6, 110.4, 101.0, 74.9, 69.4; HPLC (system B): tR = 5.0
min, purity = 99% (at 260 nm); λmax (recorded during the HPLC
analysis)/nm 258 and 382; LRMS (ESI-): m/z 320.1 [M - H]-
(100) and 641.1 [2M - H]- (70), calcd for C17H11N3O4 321.0;
2-(Benzothiazol-2-yl)-3-(4-hydroxy-2-((4-nitrobenzyl)oxy)-
phenyl)acrylonitrile (3). Benzaldehyde 2 (360 mg, 0.93 mmol,
1 equiv.) and benzothiazole-2-acetonitrile (162 mg, 0.93 mmol,
1 equiv.) were dissolved in absolute EtOH (46 mL). After, 1
drop of piperidine was added and the reaction mixture was
stirred at RT for 2 h. Thereafter, volatiles were removed under
HRMS (ESI+): m/z 344.06339 [M
+
Na]+, calcd for
C17H11N3O4Na+ 344.06418; HRMS (ESI-): m/z 320.06622 [M -
-
H]-, calcd for C17H10N3O4 320.06713.
vacuum and the resulting residue was dissolved in THF (10 Dual PLE-NTR fluorogenic "turn-on" probe (5). Under Ar
mL). The mixture was cooled to 0 °C with an ice-water bath. atmosphere, Ac-PHBA (60 mg, 0.36 mmol, 1 equiv.) was
Then TBAF (1.0 M in THF, 1 mL) was added and the mixture dissolved in dry DCM (3.6 mL). Diphosgene (60 μL, 0.504
was left to warm at RT for 40 min. Thereafter, the reaction mmol, 1. 4 equiv.) was then added and the resulting reaction
mixture was quenched by adding glacial acetic acid (0.5 mL) mixture was cooled to 0 °C with an ice bath. Then DIEA (63 μL,
and solvents were evaporated under reduced pressure. The 0.36 mmol, 1 equiv.) was added and the mixture was stirred
resulting residue was purified by chromatography on a silica again at 0 °C. Thereafter, the mixture was left to warm up to
gel column (eluent: EtOAc-MeOH-TEA, step gradient from 8 : 2 RT and stirred for a further 30 min. DCM was removed under
: 0 to 8 : 1.9 : 0.1, v/v). The product was taken with EtOAC, vacuum. At the same time, a solution of phenol 3 (50 mg, 0.12
then acidified with two drops of glacial acid acetic and washed mmol, 0.33 equiv.) in dry THF (3 mL) was prepared and cooled
with deionised water in order to remove TEA, the organic layer to 0 °C under an Ar atmosphere. The previously prepared
dried over anhydrous Na2SO4, filtered and concentrated to chloroformate derivative and TEA (47 μL, 0.35 mmol, 1 equiv.)
obtain the desired product 3 as orange solid (150 mg, yield were sequentially added and the reaction mixture was stirred
38%, mixture of E and Z isomers). Rf 0.27 (DCM-EtOAc, 9.5 : at RT overnight. Thereafter, volatiles were removed under
0.5, v/v); νmax(neat)/cm-1 3358, 2925, 2216, 1620, 1579, 1522, vacuum and the resulting residue was re-dissolved in DCM.
1367,1345, 1299; δH(300 MHz, DMSO-d6) 10.70 (bs, 1 H, OH), The solution was filtrated to remove TEA.HCl and the filtrate
8.56 (s, 1 H), 8.32 (d, J 7.8, 2 H), 8.21 (d, J 8.1, 1 H), 8.13 (d, J was evaporated to dryness. The residue was purified by
7.2, 1 H), 8.02 (d, J 7.5, 1 H), 7.78 (d, J 7.8, 2 H), 7.56-7.47 (m, 2 chromatography on a silica gel column (eluent: heptane-
H), 6.64 (m, 2 H), 5.44 (s, 2 H); δC(75 MHz, DMSO-d6) 177.4, EtOAc, step gradient from 100 to to 70 : 30, v/v) to afford the
164.5, 160.0, 153.6, 147.7, 144.8, 141.7, 134.3, 130.3, 128.6, fluorogenic "turn-on" probe 5 as yellow solid (23 mg, yield
127.5, 126.3, 124.3, 123.3, 122.9, 117.5, 113.1, 110.0, 101.3, 32%, mixture of
E and Z isomers). Rf 0.10 (DCM);
100.6, 69.4; HPLC (system B): tR = 5.8 min, purity = 100% (at νmax(neat)/cm-1 1754, 1595, 1523, 1433, 1375, 1258; δH(300
260 nm); λmax (recorded during the HPLC analysis)/nm 221, 262 MHz, CDCl3) 8.63 (s, 1 H), 8.38 (d, J 8.7, 1 H), 8.29 (d, J 8.7, 2 H),
and 395; LRMS (ESI-): m/z 428.1 [M - H]- (100), calcd for 8.08 (d, J 8.7, 1 H), 7.90 (d, J 7.2, 1 H), 7.64 (d, J 8.7, 2 H), 7.57-
C23H15N3O4S 429.1.
7.36 (m, 4 H), 7.14 (d, J 8.4, 2 H), 7.01 (dd, J 8.7, J 2.1, 1 H),
6.90 (d, J 2.1, 1 H), 5.29 (s, 2 H), 5.27 (s, 2 H), 2.32 (s, 3 H);
δC(75 MHz, CDCl3) 169.5, 157.0, 154.8, 154.3, 151.2, 143.7,
142.8, 140.2, 134.8, 132.0, 131.8, 130.0, 127.6, 127.0, 126.8,
126.6, 126.2, 124.0, 123.8, 122.0, 121.7, 119.9, 114.6, 114.0,
110.8, 105.9, 70.1, 69.7, 21.1; The complexity of the spectra
(mixture of E and Z isomers, slow relaxation rate of some
protons) for this compound makes a complete assignment
impossible particularly for minor isomer. HPLC (system A): tR =
5.9 and 6.2 min, purity = 94%; λmax (recorded during the HPLC
analysis)/nm 222 and 346 (weak, broad absorption band) for
more polar isomer, 221, 364 and 372 for less polar isomer;
LRMS (more polar isomer, ESI+): m/z 622.2 [M + H]+ (100) and
2-(4-Hydroxy-2-((4-nitrobenzyl)oxy)benzylidene)-
malononitrile (4). Benzaldehyde 2 (1 g, 2.58 mmol, 1 equiv.)
and malonitrile (180 mg, 2.84 mmol, 1.05 equiv.) were
dissolved in absolute EtOH (130 mL). After, 1 drop of
piperidine was added and the reaction mixture was stirred at
RT for 1 h. thereafter, volatiles were removed under vacuum
and the resulting residue was dissolved in THF (26 mL). The
mixture was cooled to 0 °C with an ice-water bath. Then TBAF
(1.0 M in THF, 2.85 mL) was added and the mixture was left to
warm at RT for 50 min. Thereafter, the reaction mixture was
quenched by adding glacial acetic acid (0.4 mL) and solvents
were evaporated under reduced pressure. The resulting
residue was dissolved in DCM and washed with aq. 1.0 M HCl
(20 mL) and brine (20 mL). The organic layer was dried over
anhydrous Na2SO4, filtered and concentrated under vacuum.
The resulting residue was purified by trituration in MeOH and
429.3 [M
+
H
-
C10H9O4]+ (35, loss of para-
acetoxybenzyloxycarbonyl moiety was occurred during the
ionisation process); LRMS (more polar, isomer, ESI-): m/z 428.3
[M - C10H9O4]- (100, loss of para-acetoxybenzyloxycarbonyl
moiety was occurred during the ionisation process); LRMS (less
This journal is © The Royal Society of Chemistry 20xx
Org. Biomol. Chem., 2015, 00, 1-3 | 7
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Journal Name
polar isomer, ESI+): m/z 622.1 [M + H]+ (100) and 660.7 [M + and DMAP (catalytic amount) were dissolved in dVriyewTAHrtFic.lepOanrliane-
K]+ (25); LRMS (less polar isomer, ESI-): m/z 428.3 [M - Nitrophenyl chloroformate (334 mg, 1.66 mmol, 2 equiv.) was
DOI: 10.1039/C5OB01624J
+ -
C10H9O4 ] (100, loss of para-acetoxybenzyloxycarbonyl moiety added and the resulting reaction mixture was stirred under an
was occurred during the ionisation process), calcd for Ar atmosphere for 30 min. Thereafter, EtOAc (50 mL) was
C33H23N3O8S 621.1; HRMS (ESI+): m/z 622.12739 [M + H]+, added and the organic medium was washed with aq. 1.0 M HCl
calcd for C33H24N3O8S+ 622.12786 and 644.10859 [M + Na]+, (3 × 10 mL), aq. saturated NaHCO3 (3 × 15 mL) and dried over
calcd for C33H23N3O8SNa+ 644.10981.
anhydrous MgSO4. The solvent was removed under reduced
pressure and the resulting crude product was purified by
chromatography on a silica gel column (eluent: DCM-EtOAc-
TEA 50 : 10 : 1.5, v/v/v). The activated carbonate 7 was
obtained as white solid (194 mg, yield 58%). δH(300 MHz,
CDCl3) 8.26 (d, J 9.3, 2 H), 7.50 - 7.34 (m, 11 H), 7.12 (s, 1 H,
NH), 5.22 (s, 2 H), 3.76 (s, 2 H).
Dual PLE-NTR fluorogenic "turn-on" probe (8).
(a) Preparation of para-nitrophenyl para-acetoxybenzyl
34
carbonate : Ac-PHBA (200 mg, 1.20 mmol, 1 equiv.), TEA
(0.325 mL, 2.41 mmol, 2 equiv.) were dissolved in dry THF.
para-Nitrophenyl chloroformate (485 mg, 2.41 mmol, 2 equiv.)
was added, and the resulting reaction mixture was stirred
under Ar atmosphere for 90 min. Thereafter, EtOAc (50 mL)
was added and the organic medium was washed with aq. 1.0
M HCl (3 × 10 mL), aq. saturated NaHCO3 (3 × 15 mL) and dried
over anhydrous MgSO4. The solvent was removed under
reduced pressure and the resulting crude product was purified
by chromatography on a silica gel column (eluent: DCM-EtOAc-
TEA 50 : 10 : 1.5, v/v/v). The activated carbonate 6 was
obtained as white solid (228 mg, yield 57%). δH(300 MHz,
CDCl3) 8.30 (d, J 9.3, 2 H), 7.48 (d, J 8.7, 2 H), 7,40 (d, J 9, 2 H),
7.16 (d, J 8.7, 2 H), 5.30 (s, 2 H), 2,33 (s, 3 H).
(b) Phenol protection: Under an Ar atmosphere, a solution of
activated carbonate 7 (194 mg, 0.48 mmol, 1.2 equiv.) in dry
THF (4 mL) was added to phenol 4 (128 mg, 0.398 mmol). Then
TEA (54 μL, 0.40 mmol, 1 equiv.) and DMAP (catalytic amount)
were sequentially added. The resulting reaction mixture was
stirred at RT for 1 h. Thereafter, solvent was removed under
vacuum and the resulting residue was taken with DCM and
washed with aq. 0. 1 M K2CO3 (2 × 10 mL). The organic layer
was dried over anhydrous MgSO4, filtered and concentrated
under reduced pressure. The residue was firstly purified by
chromatography on a silica gel column (eluent: heptane-
EtOAc, step gradient from 100 to 70 : 30, v/v) and then by
crystallization in pre-cooled (0 °C) mixture EtOH-acetone (8 : 2,
v/v) to obtain yellow solid (40 mg, yield 17%). Rf 0.84 (DCM-
EtOAc, 9: 1, v/v); νmax(neat)/cm-1 3289, 3064, 2229, 1767,
1657, 1605, 1521, 1361, 1229; δH(300 MHz, DMSO-d6) 10.25 (s,
1 H), 8.55 (s, 1 H), 8.28 (d, J 8.4, 2 H), 8.07 (d, J 8.7, 1 H), 7.80
(d, J 8.7, 2 H), 7.64 (d, J 8.7, 2 H), 7.41-7.12 (m, 9 H), 5.42 (s, 2
H), 5.22 (s, 2 H), 2.65(s, 2 H); δC(75 MHz, DMSO-d6) 169.7,
158.8, 156.6, 155.5, 152.4, 147.8, 143.9, 140.1, 136.4, 130.6,
129.9, 129.7, 129.6, 129.2, 128.8, 127.0, 124.1, 119.5, 118.8,
115.2, 114.7, 113.7, 107.8, 82.7, 70.7, 79.9, 43.8; HPLC (system
A): tR = 5.7 min, purity = 94% (at 260 nm); λmax (recorded
during the HPLC analysis)/nm 231, 295 and 356; LRMS (ESI-):
(b) Phenol protection: Under Ar atmosphere, a solution of
activated carbonate 6 (377 mg, 1.14 mmol, 3 equiv.) in dry THF
(4.2 mL) was added to phenol 4 (122 mg, 0.38 mmol, 1 equiv.).
Then TEA (153 μL, 1.14 mmol, 3 equiv.) and DMAP (catalytic
amount) were sequentially added. The resulting reaction
mixture was stirred at RT for 1 h. Thereafter, solvent was
removed under vacuum and the resulting residue was taken
with DCM and washed with aq. 0.1 M K2CO3 (2 × 10 mL). The
organic layer was dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure. The residue was
purified by chromatography on a silica gel column (eluent:
heptane-EtOAc, step gradient from 100 to 70 : 30, v/v) to give
the desired fluorogenic "turn-on" probe as light yellow solid
(60 mg, yield 30%). Rf 0.30 (DCM); νmax(neat)/cm-1 2956, 2924,
2228, 1762, 1604, 1589, 1518, 1435, 1357, 1245; δH(300 MHz,
CDCl3) 8.31-8.26 (m, 3 H), 8.20 (s, 1 H), 7.57 (d, J 8.7, 2 H), 7.45
(d, J 8.7, 2 H), 7.14 (d, J 8.7, 2 H), 6.53 (dd, J 9.0, J 2.4, 1 H),
6.93 (d, J 2.4, 1 H), 5.26-5.24 (2s, 4 H), 2.31 (s, 3 H); δC(75 MHz,
CDCl3) 169.3, 158.4, 156.8, 152.5, 152.2, 151.2, 148.2, 141.7,
131.7, 130.3, 130.1, 128.2, 124.3, 122.1, 118.2, 114.8, 114.0,
112.7, 105.9, 82.3, 70.3, 70.1, 21.1; HPLC (system A): tR = 5.7
min, purity = 94% (at 260 nm); λmax (recorded during the HPLC
analysis)/nm 296 and 356; LRMS (ESI-): m/z 512.3 [M - H]- (10)
m/z 320.1 [M
-
C16H14NO3]- (100, loss of para-
phenylacetamidobenzyloxycarbonyl moiety was occurred
during the ionisation process); LRMS (ESI+): m/z 589.2 [M + H]+
(100), calcd for C33H24N4O7 588.1; HRMS (ESI+): m/z 589.17148
+
[M + H]+, calcd for C33H25N4O7 589.17178 and 611.15320 [M +
Na]+, calcd for C33H24N4O7Na+ 611.15372.
In vitro activation of fluorogenic "turn-on" probes 5, 8 and 9
by hydrolase (PGA or PLE) and reductase (NTR) - Fluorescence
assays and HPLC-MS analyses
See ESI† for experimental details.
and 320.2 [M
-
C10H9O4]- (100, loss of para-
acetoxybenzyloxycarbonyl moiety was occurred during the
ionisation process), calcd for C27H19N3O8 513.1; HRMS (ESI+):
m/z 536.10566 [M + Na]+, calcd for C27H19N3O8Na+ 536.10644.
Abbreviations (not defined within the text)
BZT
calcd
CBT
2-benzothiazolyl
calculated
2-cyanobenzothiazole
Dual PGA-NTR fluorogenic "turn-on" probe (9).
CRABPII protein reengineering of cellular retinoic acid
binding protein II
(a)
Preparation
of
para-nitrophenyl
para-
35
(phenylacetamido)benzyl carbonate : PhAc-PABA (200 mg,
DCM
dichloromethane
0.829 mmol, 1 equiv.), TEA (0.223 mL, 1.66 mmol, 2 equiv.)
8 | Org. Biomol. Chem., 2015, 00, 1-3
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Page 9 of 16
Organic & Biomolecular Chemistry
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ARTICLE
DMAP
EtOH
Et2O
FA
FRET
HPLC
MS
NADH
PE
rt
4-N,N-dimethylaminopyridine
ethanol
diethyl ether
9. (a) W. Zhou, C. Andrews, J. Liu, J. W. Shultz, M.VPie.wVAartlilceleyO, nJl.inJe.
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ChemBioChem, 2008, 9, 714; (b) W. Zhou, D. Leippe,
S.
formic acid
Duellman, M. Sobol, J. Vidugiriene, M. O'Brien, J. W. Shultz, J. J.
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W. B. Porterfield, K. A. Jones, D. C. McCutcheon and J. A.
Prescher, J. Am. Chem. Soc., 2015, 137, 8656.
fluorescence resonance energy transfer
high-pressure liquid chromatography
mass spectrometry
nicotinamide adenine nucleotide (reduced form)
petroleum ether (bp 40-60 °C)
room temperature
10. R. H. F. Wong, T. Kwong, K.-H. Yau and H. Y. Au-Yeung, Chem.
Commun., 2015, 51, 4440.
TBAF
tetrabutylammonium fluoride
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TBDMS tert-butyldimethylsilyl
TBDMS-Cl tert-butyldimethylsilyl chloride
TEA
THF
triethylamine
tetrahydrofuran
ultraviolet-visible
UV-vis
Acknowledgements
Financial support from Institut Universitaire de France (IUF)
and the Burgundy region ("FABER" programme, PARI Action 6,
SSTIC 6 "Imagerie, instrumentation, chimie et applications
biomédicales"), especially for the Ph. D. grant of S. D., is
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 the
access to HORIBA Jobin Yvon Fluorolog spectrofluorimeter,
Thermo LTQ Orbitrap XL mass spectrometer, Bruker Avance III
300 and 500 spectrometers and Bruker Alpha FT-IR
spectrometer. The authors also thank Marie-José Penouilh (IE,
University of Burgundy, ICMUB-WPCM) and Dr. Fanny Picquet
(IE, ICMUB-WPCM) for recording the HRMS spectra, and
Benoît Roubinet (Ph. D. student, University of Rouen,
laboratory COBRA UMR CNRS 6014) for the synthesis of 3-(2-
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benzothiazolyl)-7-hydroxy-(2-imino)coumarins
references in this work.
used
as
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DOI: 10.1039/C5OB01624J
Organic & Biomolecular Chemistry
PAPER
P'
Y
reaction with
enzymes (2x)
Y
"covalent assembly"
Ar
Ar
Ar
Ar
Ar
Ar
in situ formation of fluorophore
X
P
X
X
X = EDG (for "push-pull" character)
but not reactive with Y
R
R
R
enzyme 1
enzyme 2
CN
CN
O
HO
O
CN
OH
P
O
HO
P'
P'
in situ formation
of 2-iminocoumarin
O
S
N
AcO
O
R = CN or
R
P = SO3H or
R = CN, Ex/Em = 418/458 nm
R = BZT, Ex/Em = 455/489 nm
or
O
NH
HO
O
P' =
O2N
O
Ph
N
O
H
enzyme 1
enzyme 2
fluorescence ("turn on" response)
Fig. 1 Strategy explored in this work: (top) "covalent assembly" principle applied to the simultaneous detection of two distinct
enzymes (X = NH or O, EDG = electron-donating group); (bottom) in situ synthesis of 3-substituted-7-hydroxy-2-iminocoumarin
scaffolds from a non-fluorescent caged precursor and through domino reactions triggered by the two different enzymes (failed
attempt: probe for sulfatase-NTR, R = CN, P = SO3H and P' = para-nitrobenzyl; probes for PLE-NTR, R = BZT or CN, P = Ac-
PHBoxycarbonyl and P' = para-nitrobenzyl; probe for PGA-NTR, R = CN, P = PhAc-PABoxycarbonyl and P' = para-nitrobenzyl). BZT
= 2-benzothiazolyl, NTR = nitroreductase, PLE = pig liver esterase and PGA = penicillin G acylase.
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View Article Online
DOI: 10.1039/C5OB01624J
O
O
O
O
Ag2O, Et2O, RT,
overnight
TBDMS-Cl, imidazole,
DCM, RT, 4 h
H
H
H
yield 72%
TBDMSO
HO
OH
TBDMSO
OH
Br
2
1
2,4-dihydroxy-
benzladehyde
O2N
NO2
yield 88%
NC
CN
1. piperidine, EtOH, RT, 1 h
2. TBAF, THF, RT, 1 h
or
S
N
CN
O
R
CN
S
O
Cl
CN
N
AcO
3, R = BZT, yield 38%
4, R = CN, yield 55%
O
HO
O
TEA, THF, 0 °C to RT
overnight
O
O
O
AcO
NO2
5
yield 32%
NO2
NO2
O
O
O
NC
CN
6, R' = AcO
7, R' = PhAc-NH
R'
O
TEA, DMAP, THF, 30-60 min
8, R' = AcO, yield 30%
9, R' = PhAc-NH, yield 17%
O
O
O
R'
NO2
Scheme 1 Synthesis of dual-enzyme fluorogenic "turn-on" probes 5, 8 and 9. Please note: molecule corresponding to
chloroformate derivative of Ac-PHBA is not numbered because not isolated and directly used without purification.
12 | Org. Biomol. Chem., 2015, 00, 1-3
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Fig. 2 Time-dependant changes in the blue/green fluorescence intensity of fluorogenic "turn-on" probes 5, 8 and 9
(concentration: 1.0 μM) in the presence of two distinct enzymes in PB at 37 °C (see ESI† for more details about sequential and
simultaneous incubations protocols with NTR (0.1 U) / NADH (45 μM), PLE (1 U) and PGA (1 U)). (A) Ex./Em. 455/489 nm; (B)-(C)
Ex./Em. 418/458 nm. For each probe, a further kinetic curve (purple trace) was recorded under the same conditions but without
enzymes and co-factor.
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Fig. 3 RP-HPLC elution profiles (fluorescence detection, system E) of enzymatic reaction mixture of benzothiazolyl-based probe 5
with PLE (10 min of pre-incubation) and NTR/NADH (A) and authentic samples of 3-(2-benzothiazolyl)-7-hydroxycoumarin (B)
and 3-(2-benzothiazolyl)-7-hydroxy-2-iminocoumarin (C). Please note: partial hydrolysis of imine moiety was occurred during
HPLC analysis and incubation in PB. NADH (tR = 3.4 min) can be properly detected at a different wavelength channel (Ex./Em.
350/460 nm).
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Fig. 4 RP-HPLC elution profiles (UV-vis and MS detection, system C) of cyano-based probe 8: (A) before incubation in PB; (B)
after 10 min of incubation in PB at 37 °C (retro-Knoevenagel product was not detected at 350 nm); (C)-(D) after dual-enzymatic
activation at 37 °C (sequential protocol, see ESI† for experimental details): (C) after 30 min of incubation with PLE (0.55 U)
(compound 4 was not detected at 350 nm); (D) after 80 min of incubation with PLE (0.55 U) alone and further 40 min with
NTR/NADH (0.6 U/0.58 mM). See figure S7 for all ESI mass spectra. See figures S8-S9 for all RP-HPLC elution profiles related to
dual-enzymatic activation of probe 8 (sequential "NTR/NADH then PLE" and simultaneous incubation protocols) and probe 9
(sequential "PGA then NTR/NADH" and "NTR/NADH then PGA" and simultaneous incubation protocols).
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Table 1 RP-HPLC retention times, LR-MS data and/or spectral properties of compounds formed through theVdieuwaAlr-tieclnezOynmlinee
activation of probes 5, 8 and 9.
DOI: 10.1039/C5OB01624J
RP-HPLC-
fluorescence
tR (min)a
RP-HPLC-
MS
Molecular ion Molecular ion
Abs./Em.
(nm)
Compound
ΦF (%)
detected
calcd
tR (min)b
S
N
5.4
5.2
-
-
-
-
446/493c
431/488d
43c
NH
HO
O
S
-
-
57d
N
O
HO
O
CN
NH
187.5
187.2
3.9
4.1
3.7
5.9
418/458e
409/452e
37f
85f
[M + H]+
[M + H]+
HO
O
CN
O
186.4
[M - H]-
186.1
[M - H]-
HO
O
CONH2
X
4.3 (X = NH) 203.2 [M - H]- 203.2 [M - H]-
3.6
-
-
-
-
-
HO
O
5.5 (X = O)
204.4 [M - H]- 204.2 [M - H]-
X = NH or O
O
O
O
H
510.1
[M - H + FA]-
510.4
[M - H + FA]-
O
O
8.2
AcO
NO2
O
O
H
584.9
585.5
O
O
O
-
-
-
8.3
7.7
7.0
-
-
-
-
-
-
[M - H + FA]-
[M - H + FA]-
PhAc
N
H
NO2
NC
CN
320.3
[M - H]-
320.3
[M - H]-
HO
O
NO2
O
O
H
272.3
[M - H]-
272.2
[M - H]-
HO
NO2
a system D. b system C. c see ref. 45. d see ref. 21. e determined in PB at 25 °C. f determined in PB at 25 °C using quinine sulfate (ΦF = 60% in
0.05 M H2SO4) as a standard.
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