In situ formation of pyronin dyes for fluorescence protease sensing

Article abstract of DOI:10.1039/c7ob00370f

We report a reaction-based strategy for the fluorogenic detection of protease activity. Based on the “covalent-assembly” probe design principle recently put forward by the Yang group for detection of Sarin related threats (J. Am. Chem. Soc., 2014, 136, 6594-6597), we have designed two unusual non-fluorescent caged precursors (mixed bis-aryl ethers) which are readily converted into a fluorescent unsymmetrical pyronin dye through a domino cyclisation-aromatisation reaction triggered by penicillin G acylase (PGA) or leucine aminopeptidase (LAP). Fluorescence-based in vitro assays and HPLC-fluorescence/-MS analyses support the claimed activation mechanism whose the further implementation to “smart” imaging agents for the study of protease function in vivo is expected.

Full text of DOI:10.1039/c7ob00370f

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ARTICLE
In situ formation of pyronin dyes for fluorescence protease
sensing†
Sylvain Debieua and Anthony Romieu*a,b
Received 00th January 20xx,
Accepted 00th January 20xx
We report a reaction-based strategy for the fluorogenic detection of protease activity. Based on the "covalent-assembly"
probe design principle recently put forward by the Yang group for detection of Sarin related threats (J. Am. Chem. Soc.,
2014, 136, 6594-6597), we have designed two unusual non-fluorescent caged precursors (mixed bis-aryl ethers) which are
readily converted into a fluorescent unsymmetrical pyronin dye through a domino cyclisation-aromatisation reaction
triggered by penicillin G acylase (PGA) or leucine aminopeptidase (LAP). Fluorescence-based in vitro assays and HPLC-
fluorescence/-MS analyses support the claimed activation mechanism whose the further implementation to "smart"
DOI: 10.1039/x0xx00000x
www.rsc.org/
imaging
agents
for
the
study
of
protease
function
in
vivo
is
expected.
3
target analyte. The analyte-mediated synthesis of blue-green
Introduction
emitting 7-N,N-dialkylamino/7-hydroxy (2-imino)coumarins is
4
often preferred for in vitro (bio)sensing applications but was
5
During the past decade, reaction-based small-molecule
fluorescent probes (also known as fluorescent
also implemented in photoaffinity labeling experiments. We
have recently used this fluorogenic cyclisation reaction for
designing dual-input fluorogenic probes (acting as AND
molecular logic gates) dedicated to simultaneous detection of
chemodosimeters, latent or pro-fluorophores) have been
widely adopted as valuable (bio)analytical tools for the
detection and/or imaging of various (bio)analytes including
enzymes, biomolecules, polluting chemicals, cations and
two distinct enzyme activities namely hydrolase and
6
reductase. Furthermore, the Yang group has successfully
anions, in complex biological systems or environmental
1
applied the "covalent-assembly" principle to longer-
wavelength fluorescent scaffolds including pyronin B (Fig. 1),
phenanthridine, benzo[c]cinnoline and resorufin derivatives,
matrices. The majority of these chemodosimeters are
rationally constructed through the reversible protection of a
key functional group of an organic-based fluorophore
7
8
for the detection of Sarin mimics , Hg(II) cation , MeHg(I)
10
(typically, an amino or hydroxyl group of a coumarin or
2
9
cation and reactive nitrogen species (RNS)
respectively.
xanthene dye). Despite its many successes, this approach
However, the majority of these latter probes suffers from a
lack of versatility (the chemical cascade is specific to the target
analyte and its inherent reactivity and cannot be triggered by
an other analyte) preventing (1) their use for sensing a wide
range of analytes including enzymes and biomolecules, and (2)
may sometimes lead to unsatisfactory results in terms of
selectivity and sensitivity toward the target analyte. This is the
consequence of the poor stability of some pro-fluorophores in
aq. biological media (some aniline- or phenol-based
fluorophores are good leaving groups) and/or their high
background fluorescence due to incomplete quenching of the
masked fluorophore. In order to remedy these drawbacks, a
novel class of reactive fluorescence "turn-on" probes based on
the "covalent-assembly" principle has recently emerged. This
approach is based on in situ formation of an organic-based
fluorophore from a caged bifunctional precursor and through
effective (biocompatible) domino reactions, triggered by the
a
possible further extension of the "covalent-assembly"
11, 12
approach to dual- or multi-analyte detection schemes.
In
order to respond to these challenges, we have decided to
investigate in situ formation of pyronin dyes triggered by
enzymatic events. Such xanthene-based fluorophores have
been selected because their spectral features in the yellow-
orange region will be valuable for effective biosensing in
complex biological media. Furthermore, the upstream design
of pyronin precursors where the two aniline moieties are
orthogonally protected will open the way for the next
13
construction of dual-input fluorogenic probes.
In this paper, we report our progress toward the
development of an unprecedented class of protease-sensing
"smart" fluorescent probes whose the principle mechanism of
activation is based on the pyronin assembly triggered by the
enzyme itself (Fig. 1).
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at 90 °C. At higher temperatures, significant degradation of
starting materials and targeted coupling product was
observed. Finally, removal of the Boc group of crude coupling
product was achieved under anhydrous acidic conditions (60%
TFA in DCM) at 0 °C for 20 min. After removal of TFA under
mild conditions (essential to avoid side-reactions leading to
degradation and/or premature formation of pyronin
derivatives), purification was achieved by semi-preparative RP-
Results and discussion
To rapidly establish a proof-of-concept, we have chosen to
work with two distinct hydrolases namely penicillin G acylase
(PGA, also known as penicillin amidase) and leucine
aminopeptidase (LAP from porcine kidney) that have two clear
advantages:
(1)
structurally
simple
substrates
(phenylacetamide and L-leucine amide moieties respectively)
than can be easily installed on the pyronin precursor through
amidification of its primary aniline and (2) a commercial
availability at low or reasonable cost. PGA is widely used as
HPLC to give the TFA salt of LAP-sensitive fluorogenic probe
in a low but not optimized yield (11% overall yield for the last
two steps). All spectroscopic data (see ESI ), especially IR,
5
†
biocatalyst in the synthesis of β-lactam antibiotics, since it
allows for the deprotection of phenylacetyl-protected
NMR and mass spectrometry, were in agreement with the
structures assigned. Their purity was checked by RP-HPLC and
14
amines , and often regarded as a valuable of model protease
found to be 99% (
these syntheses, we have also prepared an authentic sample
of unsymmetrical pyronin (6-N,N-diethylamino-3H-xanthen-
4) and 93% (5) respectively. In addition to
to perform in vitro validations of self-immolative molecular
systems used as diagnostic probes, molecular amplifiers or
15
6
drug delivery systems.
16
LAP is a metallopeptidase widely
3-imine, see Fig. 1 for the corresponding structure) which will
be formed upon enzymatic activation of the two probes, and
used as reference compound for the fluorescence-based in
vitro assays and HPLC-fluorescence/-MS analyses (vide infra).
This unsymmetrical xanthene-based fluorophore has never
been described in the literature but a recent publication from
the Lin group about the synthesis of a 7-hydroxycoumarin-
studied as an attractive drug target in anti-cancer and anti-
17
malaria chemotherapies. Furthermore, placental LAP (P-LAP)
was recently identified as a relevant biomarker for predicting
18
perinatal mortality (e.g., serum P-LAP levels are extremely
low among women with foetal death). Interestingly, some self-
immolative fluorogenic reporters for LAP sensing in living cells
19
have been recently developed by the Hasserodt group.
pyronin FRET dyad and its use as a ratiometric fluorescent
22
Inspired by the nerve agent detection mechanism reported
7
probe for H2S sensing , helped us to devise a synthesis of this
by Lei and Yang for probe NA570 , we assumed that a quite
unusual pyronin from 4-(N,N-diethylamino)salicylaldehyde and
3-aminophenol (see ESI†).
similar bis-aryl ether derivative for which one of the N,N-
diethylamino group is replaced by a N-acyl primay amino
moiety, may act as a fully stable non-fluorescent pyronin
precursor. Since the selective enzymatic deprotection of its
carboxamide group generates an intermediate that will
spontaneously cyclise through intramolecular reaction
between the unveiled electron-rich arene moiety and
All UV-visible and fluorescence measurements were
conducted in phosphate buffer (PB, 100 mM, pH 7.6, simulated
physological conditions) containing less than 1% of CH3CN as a
co-solvent and the corresponding data are gathered in Table 1
(see Fig. S1-S3 for the corresponding spectral curves). The two
protease-sensitive probes
absorption in the UV-A range and are not fluorescent,
especially in the expected emission range of pyronin (500-
4 and 5, exhibits a strong electronic
aldehyde to give, after aromatisation
a
fluorescent
unsymmetrical pyronin dye (Fig. 1). Interestingly, the multiple
sources of primary amines in the reaction medium (i.e., lysine
residues within the active site of protease, released L-leucine
6
600 nm), whatever the excitation wavelength used. To
demonstrate the feasability of proposed detection mechanism,
and/or primary aniline of intermediate) should lead to in situ
20
we also investigated the photophysical properties of pyronin
in PB.
6
formation of a more reactive imine/iminium intermediate
and favor the cyclisation process in this neutral aq.
environment.
Table 1 Photophysical properties of the pobes 4 and 5 and pyronin dye
in phosphate buffer (100 mM, pH 7.6) and at 25 °C.
6
The synthesis of the two protease-sensitive fluorogenic
probes
4 and 5 was achieved in two or three steps as depicted
Abs. max
(nm)
Em. max
(nm)
ε
Cmpd
Φ
F (%)a
in Scheme 1. First, 3-iodoaniline was N-acylated either with
commercial phenylacetyl chloride (PhAc-Cl) or mixed
anhydride from Boc-L-leucine and isobutyl chloroformate
(M-1 cm-1)
14 860,
16 040
b
b
4
264, 368
-
-
(IBCF), to give compounds
respectively). The key step leading to unsymmetrical bis-aryl
ether scaffold is an Ullman ether synthesis from phenol and
aryl iodide or , performed under conditions previously
2 and 3 in good yields (90% and 70%
19 200,
39 800
b
b
5
6
250, 356
527
-
-
1
70 350
544
7
2
3
adetermined in PBS at 25 °C using rhodamine 6G (
Φ
F = 92% in water, Ex.
21
optimized by the Buchwald group.
The PGA-sensitive
23
470 nm) as a standard
.
bnon-fluorescent.
fluorogenic probe 4 was readily formed on heating reaction
mixture at 130 °C for 12 h and isolated in a pure form by
conventional flash-column chromatography on silica gel.
Conversely, in the case of LAP-sensitive probe, this copper-
catalysed coupling did not work properly (checking by periodic
HPLC-MS monitoring) when the reaction mixture was heated
The UV-visible absorption spectrum of 6 displays a maximum
= 70 350 M-1 cm-1) and the emission spectrum
at 527 nm (
ε
shows a peak maximum at 544 nm (ΦF = 7%). Interestingly, a
perfect matching between the absorption and excitation
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A
spectra is observed (see Fig. S2), supporting the lack of H-type
aggregates under simulated physiological conditions. These
photophysical studies show that the enzymatic cleavage of
fluorogenic probes
4 and 5 and subsequent cyclisation-
aromatisation process would produce a strong green-yellow
fluorescence signal centered at 545 nm.
Fluorescence-based in vitro assays were performed with
commercial enzymes (PGA from Escherichia coli and LAP
microsomal from porcine kidney) through time-course
measurements (Fig. 2A and Fig. S4-S9). The first tests were
conducted on the LAP-sensitive probe
5 and had confirmed
B
our starting hypothesis related to in situ pyronin formation
triggered by protease. Indeed, a significant and gradual
increase of green-yellow fluorescence intensity at 545 nm (Ex.
525 nm) was observed when
5 was incubated with LAP at 37 °C
for 14 h. In contrast, without addition of LAP, probe
5
displayed minor fluorescence change under the same
conditions (diminished by 17-fold compared to the
fluorescence level reached with LAP). The slight instability of
5
at physiological pH may well stem from hydrolysis of its
anilinamide bond, more reactive than an alkyl counterpart.
This non-specific fluorescence signal could be completely Fig. 2 (A) Time-dependant changes in the green-yellow fluorescence
24
intensity (Ex./Em. 525/545 nm, bandwidth 5 nm) of fluorogenic probe
suppressed by implementing a dual-reactable strategy. In an
5
(concentration: 1.0 ꢀ
M) in the presence of LAP (2 x 10-3 U solid lines
attempt to accelerate the fluorogenic activation kinetics of
probe , two other aq. buffers at acidic and basic pH
and 10-2 U for dotted line) in aq. buffers at 37 °C; (B) Fluorescence
emission spectra (Ex. 525 nm, bandwidth 5 nm) of selected enzymatic
reaction mixtures after 24 h of incubation at 37 °C and authentic
5
respectively (acetate buffer, 100 mM, pH 5.6 and borate
buffer, 100 mM, pH 8.6) were assessed. In both cases, a lower
fluorescence end-point value was obtained than that achieved
in PB, suggesting the less efficiency of LAP at acidic pH and the
ineffectiveness of cylisation at basic pH. However, the shape of
kinetic curve obtained at pH 5.6 has drawn our attention
specifically. Indeed, a curved line was observed that can be
interpreted as follows : over a period of time of ca. 2 h, the low
slope of the curve was not constant and slowly/gradually
sample of pyronin
blue-shifted emission curve obtained for reaction with LAP in PB at 37
°C was explained by the formation of 6-N-ethylamino-3H-xanthen-3-
imine resulting from dealkylation of in situ generated pyronin 6.
6 at the same concentration (1.0 ꢀM). Please note:
Considering the more robustness of the PGA enzyme
26
(compared to LAP) under these mild acidic conditions
,
further tests on the PGA-sensitive probe 4 were then
performed to validate this hypothesis (see Fig. S4, S5 and S9).
The results were clear, at acidic pH a better fluorogenic
response was achieved which indicated a positive action of
protons in the mechanism leading to the formation of the
fluorescent pyronin core after enzymatic cleavage of the
amide bond. However, a new interrogation had emerged
concerning the lower levels of fluorescence reached upon
enzymatic activation of the PGA-sensitive probe compared to
LAP-sensitive probe. To clarify this point, additives found in the
commercial sample of LAP (ammonium sulfate and magnesium
chloride) were added to PB separately or together, and
enzymatic kinetics with PGA were performed. These further
analyses clearly highlighted the catalytic role of (NH4)2SO4 in
increased, suggesting a less effective hydrolysis of
5
by the
25
protease (LAP is known to be less efficient at this tested pH)
.
Thereafter, the slope was constant and the fastest kinetics
was in agreement with the positive impact of acidic pH on
cyclisation/aromatisation domino process.
the cyclisation reaction (probably as a source of ammonia to
20
generate more reactive imine intermediate) unlike MgCl2
which seemed to have no impact. Such data could suggest a
mechanism involving an imine formation (propably protonated
at physiological pH) to favor the cyclisation process. Finally,
blank experiments without enzyme were performed to ensure
that (NH4)2SO4 or MgCl2 did not induced the generation of the
pyronin dye, and so corroborate that protease is required to
obtain a significant fluorescence response. To confirm that the
resulting fluorescence emission observed during these time-
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course measurements was due to 6-N,N-diethylamino-3H- generation of fluorescent pyronin dye. All these results
xanthen-3-imine dye formed in situ (and to clarify the reason allowed us to propose the sensing mechanism depicted in Fig.
for the blue-shifted shoulder observed in most fluorescence 4 and that could explain the slow release of pyronin core, the
emission spectra recorded after enzymatic kinetics, see Fig. pH effect and the positive impact of additive (NH4)2SO4,
2B), some enzymatic reaction mixtures were analysed by RP- leading to the significant fluorescence "turn-on" response of
HPLC coupled with fluorescence detection and synthesised probes
pyronin was used as reference. These chromatographic
analyses have confirmed the formation of pyronin dye and
4 and 5 to proteases.
6
6
clearly revealed the presence of a second fluorescent species
in the crude enzymatic reaction which needed to be identified
(see Fig. 3 and Fig. S10-S12). Finally, the specificity of the
enzymatic cleavage was studied by conducting two control
reactions : incubation of PGA-sensitive probe
incubation of LAP-sensitive probe with PGA respectively. As
expected, no significant increase of green-yellow fluorescence
at 545 nm (Ex. 525 nm) was observed when was incubated
4 with LAP and
5
4
with LAP at 37 °C for 14 h (Fig. S13), confirming that this latter
protease is not able to cleave the phenylacetamide moiety.
Conversely and quite surprisingly, PGA was able to readily
activate the LAP fluorogenic substrate
5 because a time-
dependant fluorescence response similar to that obtained with
LAP was observed (Fig. S14). Further work is currently in
progress to gain insights about this novel reactivity of bacterial
PGA, already reported for other acylamidases for which aryl
amides such as acide para-nitroanilides (especially, Leu-pNA)
27
are good substrates.
To achieve this and to gain a better understanding of the
sensing mechanism, further enzymatic reactions were
performed (at a higher concentration than this used for
fluorescence-based assays, 80
ꢀM against 1.0 ꢀM) and
periodically analysed by HPLC-MS (see Fig. S15-S17). The first
clarification provided by these analyses has concerned the
unknown fluorescent species previously noted (vide supra).
The peak assigned to this compound (tR = 3.4 min) was
identified as 6-N-ethylamino-3H-xanthen-3-imine (MS(ESI+):
m/z = 238.8 [M + H]+, calcd for C15H15N2O+ 239.1 and UV-vis:
λmax = 510 nm). Furthermore, its spectral features (absorption
and fluorescence emission maxima) were changed (compared
Fig.
3 RP-HPLC elution profiles (fluorescence detection, system H) of
enzymatic reaction mixture of probe 5 with LAP (24 h of incubation in PB at
37 °C) (A), authentic sample of pyronin 6 (B) and probe 5 without LAP (24 h
of incubation in PB at 37 °C) (C). Please note: for all analyses, concentration
of injected solution: 1.0 ꢀM and injection volume: 10 ꢀL. Ratio between
fluorecence signal detected for enzymatic reaction (A) and control without
LAP (C) is ca. 100.
to N,N-diethyl pyronin
6
) in a similar way than already
28
reported for rhodamine dyes. At this stage of the study, the
rationale for this N-dealkylation process in aq. medium
remains unknown. The second valuable information disclosed
by the HPLC-MS analyses has concerned the visualisation (tR =
4.3 min) and unambiguous identification of key intermediate
Conclusions
For the first time, we have demonstrated that the "covalent-
assembly" probe design principle could be successfully applied
to protease sensing in the spectral range 500-600 nm, through
in situ generation of unsymmetrical pyronin scaffold. This
novel class of activatable fluorescent probes could be a
valuable alternative to more conventional FRET-based (or self-
quenched) probes and fluorogenic substrates derived from
fluorescent anilines (typically pro-fluorophores derived from 7-
namely
2-(3-aminophenoxy)-4-N,N-
(diethylamino)benzaldehyde (MS(ESI+): m/z = 285.4 [M + H]+,
+
calcd for C17H21N2O2 285.1 and UV: λmax = 353 nm) which
resulted from the enzymatic hydrolysis of the amide bond of
probes
the same molecular weight but it was excluded because the
chemical equilibrium with the corresponding pyronin is
4 and 5. The cyclised xanthydrol derivative (ROH) has
6
aminocoumarin, rhodamine 110, cresyl violet and related
30
dramatically favored under acidic conditions used for these RP-
benzophenoxazines, ...)
even if the reaction rate of
HPLC analyses (0.1% formic acid in water and CH3CN, pH
29
cyclisation/aromatisation domino process should be increased.
As previously achieved with the rhodamine-based
fluorophores, the one-atom replacement of the bridging
oxygen atom of caged pyronin precursors by a quaternary
3.0). Also worthy of note is that no cyclic product N-acylated
with the enzyme-labile moiety was detected, thus confirming
the need of a protease-mediated cleavage event to trigger the
cyclisation/aromatisation domino process leading to in situ
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31
32,33
(EZ50150, 841 U/mL) and microsomal LAP (from porcine kidney)
was supplied by Sigma-Aldrich (L5006, Type IV-S, ammonium sulfate
suspension, 10-40 U/mg protein).
carbon
or silicon atom,
a pentavalent phosphorus
34
atom (alkyl(diaryl)phosphine oxide or phosphinate moiety)
35
or
a
sulfone group
should be an effective way to
dramatically shift absorption/emission maxima of in situ
generated pyronin dye from visible to far-red/NIR region (Fig.
5, top). This will facilitate in vivo molecular imaging of enzyme
activity. Further extension of this promising concept to the
construction of two-input fluorogenic probes suitable for dual-
analyte detection, is currently in progress in our laboratory
(Fig. 5, bottom). Indeed, the ability of a fluorescent probe to
monitor simultaneously two or more biomarkers for one
Instrument and methods
Lyophilisation steps were performed with an Christ Alpha 2-4 LD
plus. Centrifugation steps were performed with a Thermo Scientific
Espresso Personal Microcentrifuge instrument. 1H-, 13C- and 19F-
NMR spectra 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 solvent
pathology is particularly attractive for improving medical
12, 36
37
signal. J values are expressed in Hz. IR spectra were recorded
diagnostics.
with a Bruker Alpha FT-IR spectrometer equipped with an universal
ATR sampling accessory or with
a Bruker IR-FT Vector 22
H
R
N
X
NEt2
X = C(CH3)2, Si(CH3)2,
P(O)R', P(O)OR' or SO2
spectrometer equipped with an ATR "Golden Gate" with diamond
crystal. The bond vibration frequencies are expressed in reciprocal
centimeters (cm-1). Elemental analyses (C, H, N, S) were perfomed
on a Thermo Scientific Flash EA 1112 instrument. Optical rotation
was measured on a PerkinElmer Polarimeter Model 341 with the
O
O
Far-red/NIR "covalent assembly" probes
for molecular imaging of proteases
H
sodium D-line (589 nm) at 20 °C and [α
]D values are given in dm-1 g-1
P
N
O
O
X P'
X = NH or O
cm3. 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 ESI
source. UV-visible spectra were obtained on a Jasco V-630 Bio
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 Jasco FP-8500
spectrofluorometer (software Spectra Manager) with a standard
fluorometer cell (Labbox, LB Q, 10 mm). Emission spectra were
recorded under the same conditions after excitation at the
corresponding wavelength (Ex/Em bandwidth = 5 nm, response = 1
s and PMT sensitivity = medium). All fluorescence spectra were
corrected.
P = carboxamide or self-immolative carbamate moiety
P' = self-immolative carbamate/carbonate moiety
Two-input fluorogenic probes
for dual-analyte detection
Fig. 5 Possible ways to apply the "covalent-assembly" principle to
molecular imaging of proteases (top) and dual-analyte detection
(bottom).
Experimental section†
For the preparation of starting benzaldehyde
1 and
experimental details related to fluorescence-based in vitro
assays and HPLC-fluorescence/-MS analyses, see ESI†.
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
Fluorescence quantum yield of 6-N,N-diethylamino-3H-xanthen-3-
performed on silica gel (63-200 µm) from Sigma-Aldrich (technical
imine 6 (TFA salt) was measured in PBS at 25 °C by a relative
23
grade) or on automated flash chromatography purification system
(Interchim puriFlash™ 430). THF and DCM (HPLC-grade) were dried
over alumina cartridges using a solvent purification system PureSolv
method using rhodamine 6G (ΦF = 92% in H2O) as a standard (for
the corresponding Abs/Ex/Em spectra, see Fig. S2). The following
equation was used to determine the relative fluorescence quantum
PS-MD-5 model from Innovative Technology. TEA was stored over yield:
anhydrous Na2SO4. Anhydrous DMSO and DMF were purchased
from Carlo Erba, and stored over 3 Å molecular sieves. The HPLC-
gradient grade acetonitrile (CH3CN) was obtained from Carlo Erba.
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
Formic acid (FA, grade "eluent additive for LC-MS") was provided
by Sigma-Aldrich. Phosphate buffered saline (PBS, 100 mM + 150
mM NaCl, PH 7.5), phosphate buffer (PB, 100 mM, pH 7.6), acetate
buffer (NaOAc, 100 mM, pH 5.6), borate buffer (100 mM, pH 8.6)
and aq. mobile-phases for HPLC were prepared using water purified
with a PURELAB Ultra system from ELGA (purified to 18.2 Mꢁ.cm).
PGA (from Escherichia coli) was provided by Iris Biotech GmbH
subscripts
s and x represent standard and unknown,
respectively. The following refractive index values are used:
1.333 for H2O and 1.337 for PBS.
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Organic & Biomolecular Chemistry
Automated flash-column chromatography and high-performance
liquid chromatography separations
3-(Phenylacetamide)iodobenzene (2). To a solution of phenylacetic
acid (1.5 g, 11.0 mmol, 1 equiv.) in dry DCM (10 mL) was added
SOCl2 (1.20 mL, 16.5 mmol, 1.5 equiv.) and dry DMF (84 μL, 1.1
mmol, 0.1 equiv.) and the resulting reaction mixture stirred at RT
for 1 h. Thereafter, solvent was removed in vacuo to give PhAcCl
which was kept under an Ar athmosphere and then slowly added to
a mixture of 3-iodoaniline (2 g, 9.13 mmol, 0.8 equiv.) and TEA (1.53
mL, 11.0 mmol, 1 equiv.) in dry THF (35 mL) at 0 °C under an Ar
athmosphere. The reaction mixture was strirred for 2 h 30 between
0 °C and RT and then concentrated under reduced pressure. The
residue was retaken in EtOAc, washed with deionised water, aq. 5%
K2CO3 and brine and then dried over anhydrous MgSO4, filtered,
and concentrated to afford the product 2 as brown solid (2.85 g,
Several chromatographic systems were used for the analytical
experiments (HPLC-MS or HPLC-fluorescence) and the
purification
steps
respectively:
System A:
RP-HPLC
(Phenomenex Kinetex C18 column, 2.6 µm, 2.1 × 50 mm) with
CH3CN (+ 0.1% FA) and 0.1% aq. formic acid (aq. FA, pH 2.7) as
eluents [5% CH3CN (0.1 min) followed by linear gradient from
5% to 100% (5 min) of CH3CN] at a flow rate of 0.5 mL min-1.
UV-visible detection was achieved at 220, 260, 320 and 540
nm (+ diode array detection in the range 220-700 nm). Low
resolution ESI-MS detection in the positive/negative mode (full
scan, 100-700 a.m.u., data type: centroid, needle voltage: 3.0
kV, probe temperature: 350 °C, cone voltage: 75 V and scan
time: 1 s). System B: System A with UV-visible detection at 220,
260, 500 and 550 nm (+ diode array detection in the range
220-800 nm). Low resolution ESI-MS detection in the
positive/negative mode (full scan, 150-1500 a.m.u.). System C:
semi-preparative RP-HPLC (Thermo BetaBasic-C18 column, 5
μm, 150 × 30 mm) with CH3CN and H2O as eluents [40% CH3CN
(5 min), followed by a gradient of 40% to 55% CH3CN (15 min),
then 55% to 80% CH3CN (35 min)] at a flow rate of 20.0 mL
min-1. Dual UV detection was achieved at 260 and 350 nm.
System D: semi-preparative RP-HPLC (Thermo Betabasic-
C18 column, 5 μm, 150 × 30 mm) with CH3CN and H2O as
eluents [30% CH3CN (5 min), followed by a gradient of 30% to
60% CH3CN (20 min), then 60% to 95% CH3CN (35 min)] at a
yield 90%). IR (ATR):
1395, 1343, 1281, 1238, 1192, 1173, 1090, 1060, 1028, 994, 974,
881, 861, 768, 716 cm-1; 1H NMR (300 MHz, DMSO-d6):
= 3.63 (s, 2
ν = 3249, 3026, 2918, 1653, 1575, 1518, 1470,
δ
H, CH2-PhAc), 7.10 (t, J = 8 Hz, 1 H, H-Ar), 7.23-7.34 (m, 5 H, H-
PhAc), 7.40 (bd, J = 7.8 Hz, 1 H, H-Ar), 7.53 (bd, J = 8.1 Hz, 1 H, H-Ar),
8.13 (t, J = 1.8 Hz, 1 H, H-Ar), 10.26 (s, 1 H, NH) ppm; 13C NMR (126
MHz, DMSO-d6):
δ = 43.3, 94.5, 118.2, 126.6, 127.3, 128.3 (2 C),
129.1 (2 C), 130.8, 131.7, 135.6, 140.6, 169.3 ppm; HPLC (system A):
tR = 4.9 min (purity 93% at 260 nm); LRMS (ESI+, recorded during
RP-HPLC analysis): m/z 338.0 [M + H]+ (60) and 379.0 [M + H +
CH3CN]+ (100), calcd for C14H13INO+ 338.0; LRMS (ESI-, recorded
during RP-HPLC analysis): m/z 335.9 [M - H]- (100) and 382.1 [M - H
+ FA]- (25), calcd for C14H11INO- 336.0.
flow rate of 20.0 mL min-1. Dual UV detection was achieved Boc-
at 260 and 350 nm. System E: semi-preparative RP-HPLC Boc-
L
-leucine meta-iodophenylamide (3). To a stirred solution of
L
-leucine (1.5 g, 6.45 mmol, 1 equiv.) in dry THF (20 mL) at -20
(SiliCycle SiliaChrom C18 column, 10 μm, 20 × 250 mm) with °C, N-Methylmorpholine (NMM, 715 µL, 6.45 mmol, 1 equiv.) and
CH3CN and aq. 0.1% TFA (pH 2.0) as eluents [0% CH3CN (5 isobutyl chloroformate (840 µL, 6.45 mmol, equiv.) were
1
min), followed by a gradient of 0% to 15% CH3CN (10 min), successively added. After an activation period of 25 min, 3-
then 15% to 60% CH3CN (60 min)] at a flow rate of 20.0 mL iodoaniline (1.835 g, 6.45 mmol, 1.3 equiv.) in dry THF (5 mL) was
min-1. Quadruple UV-vis detection was achieved at 280, 350, slowly added to the above solution. The reaction mixture was
425 and 530 nm. System F: semi-preparative RP-HPLC (Thermo stirred for 3 h between -5 °C and 0 °C. Thereafter, the resulting
Hypersil GOLD C18 column, 5 μm, 10 × 250 mm) with CH3CN solution was quenched by adding 10 mL of aq. saturated NaHCO3
and aq. 0.1% TFA (pH 2.0) as eluents [0% CH3CN (5 min), and then was extracted with DCM (3 × 50 mL). The organic layers
followed by a gradient of 0% to 15% CH3CN (10 min), then 15% were combined and washed with aq. 1 N HCl and brine, dried over
to 75% CH3CN (60 min)] at a flow rate of 4.0 mL min-1. anhydrous MgSO4, filtered, and concentrated to afford the product
Quadruple UV-vis detection was achieved at 280, 350, 425 and 3 as white solid (1.946 g, yield 70%). IR (ATR):
530 nm. System G: System A with triple UV-visible detection 1586, 1526, 1472, 1416, 1391, 1365, 1289, 1244, 1160, 1047, 1023,
which was achieved at 260, 350 and 525 nm (+ diode array 993, 877, 773 cm-1; 1H NMR (300 MHz, DMSO-d6):
= 0.88 (m, 6 H,
ν = 3284, 2957, 1663,
δ
detection in the range 220-700 nm). System H: RP-HPLC- CH3-Leu), 1.20-1.75 (m, 12 H, CH3-Boc and CH2-CH-Leu), 4.08 (m, 1
fluorescence (Phenomenex Kinetex C18 column, 2.6 µm, 2.1 × H, CH-NH-Leu), 7.09 (m, 2 H, H-Ar), 7.39 (d, J = 8.4 Hz, 1 H, H-Ar),
50 mm) with same eluents and gradient as System A. 7.55 (d, J = 8.1 Hz, 1 H, H-Ar), 8.11 (bs, 1 H, NH-Boc), 10.05 (s, 1 H,
Fluorescence detection was achieved at 45 °C at the following NH) ppm; 13C NMR (126 MHz, DMSO-d6):
Ex./Em. channels: 525/545 nm and 510/530 nm (sensitivity: 1, (3 C, CH3-Boc), 40.4, 53.5, 78.0, 94.4, 118.4, 127.4, 130.7, 131.7,
PMT 1, filter wheel: auto). 140.4, 155.4, 172.0 ppm; HPLC (system A): tR = 5.4 min (purity 99%
δ = 21.5, 22.9, 24.3, 28.2
One chromatographic system was used for the purification on at 260 nm); LRMS (ESI+, recorded during RP-HPLC analysis): m/z
an automated flash chromatography purification system 433.1 [M + H]+ (60) and 376.7 [M - tBu + H]+ (100), calcd for
+
(Interchim puriFlash™ 430): System I: RP-C18 cartridge (SiliCycle C17H26IN2O3 433.1; LRMS (ESI-, recorded during RP-HPLC analysis):
SiliaSep™ C18, 40 g) with CH3CN and aq. 0.1% TFA (pH 2.0) as m/z 430.9 [M - H]- (70) and 476.9 [M - H + FA]-(45), calcd for
eluents [0% CH3CN (1.5 CV), followed by a gradient of 0% to C17H24IN2O3- 431.1.
10% CH3CN (2 CV), then 10% to 70% CH3CN (10 CV)] at a flow
rate of 26 mL min-1. Triple UV-vis detection was achieved
PGA-sensitive probe (4). A mixture of benzaldehyde 1 (400 mg,
2.07 mmol, 1.2 equiv.), iodoaryl derivative 2 (582 mg, 1.73 mmol, 1
at 250, 500 and 550 nm.
equiv.), finely ground K3PO4 (732 mg, 3.45 mmol, 2 equiv.), CuI (33
6 | Org. Biomol. Chem., 2017, 00, 1-3
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mg, 0.17 mmol, 0.1 equiv.) and picolinic acid (43 mg, 0.35 mmol, 0.2 signal), 44.2 (2 C, CH2-Et), 51.8, 101.1, 108.0, 108.2, 113.4, 114.1,
equiv.) in dry DMSO (4.2 mL) was heated in a sealed tube at 130 °C 115.3, 130.3, 130.4, 139.5, 153.4, 158.0, 160.0, 168.0, 185.2 ppm;
overnight. The reaction was checked for completion by HPLC 19F NMR (470 MHz, DMSO-d6):
δ = -73.7 (s, 3 F, CF3-TFA); HPLC
(system A) and quenched with 10 mL of deionized water. Then, the (system A): tR = 4.0 min (purity 93% at 260 nm); LRMS (ESI+,
resuting mixture was extracted with EtOAc, washed with brine, recorded during RP-HPLC analysis): m/z 398.1 [M + H]+ (100), calcd
+
dried over anhydrous Na2SO4, filtered and concentrated under for C23H32N3O3 398.2; LRMS (ESI-, recorded during RP-HPLC
reduced pressure. The residue was purified by flash-column analysis): m/z 395.9 [M - H]- (100) and 442.1 [M + H - FA]- (40) , calcd
-
chromatography over silica gel (cartridge 40 g) using an automated for C23H30N3O3 396.2; HRMS (ESI+): m/z 398.24329 [M + H]+, calcd
+
purification system (Interchim puriFlash™ 430), eluted with a linear for C23H32N3O3 398.24382 and 420.22500 [M + Na]+, calcd for
gradient of EtOAc in DCM (from 0% to 40%) to get bis-aryl ether 4 C23H31N3O3Na+ 420.22576; Elemental anal.: Found C, 57.1; H, 6.5;
as brown solid (380 mg, yield 54%). Please note: A second N, 7.8. C23H31N3O3 . 1.2 CF3CO2H requires C, 57.1; H, 6.1; N, 7.9%;
purification by semi-preparative HPLC (system C, tR = 28.0-30.9 min) UV-vis: λmax(PBS)/nm 250 and 356 (ε/dm3 mol-1 cm-1 19 200 and 39
can afford a pale yellow solid after freeze-drying. IR (ATR):
ν = 3289, 800); [α]D +39 ° (c 0.2 in CH3OH).
2971, 1664, 1590, 1522, 1484, 1436, 1406, 1393, 1353, 1262, 1196,
1149, 1097, 1076, 1001, 983, 869, 778, 719 cm-1; 1H NMR (300 MHz,
6-N,N-Diethylamino-3H-xanthen-3-imine (6).
A
mixture of
benzaldehyde 1 (100 mg, 0.52 mmol, 1 equiv.) and 3-aminophenol
(59 mg, 0.54 mmol, 1 equiv.) in TFA (3.35 mL) was heated under
reflux for 24 h. The reaction was checked for completion by HPLC
(system A). To favor dehydration process (leading to desired
pyronin), methanesulfonic acid (MsOH, 0.5 mL) was added and the
DMSO-d6):
δ = 1.04 (t, J = 7.0 Hz, 6 H, CH3-Et), 3.34 (mask by H2O
signal) (q, J = 7.0 Hz, 4 H, CH2-Et), 3.61 (s, 2 H, CH2-PhAc), 6.11 (d, J =
2.4 Hz, 1 H, H-Ar), 6.60 (dd, J = 2.1 Hz, J = 9.0 Hz, 1 H, H-Ar), 6.74
(dq, J = 1.5 Hz, J = 2.1 Hz, J = 7.8 Hz, 1 H, H-Ar), 7.22-7.40 (m, 8 H, H-
Ar and H-PhAc), 7.65 (d, J = 9.0 Hz, 1 H, H-Ar), 9.89 (s, 1 H, H-
reaction mixture was stirred under reflux for further
6 h.
formyl), 10.26 (s, 1 H, NH) ppm; 13C NMR (126 MHz, DMSO-d6):
δ =
Thereafter, the mixture was evaporated under reduced pressure.
The resulting residue was dissolved in a mixture of aq. 0.1% TFA and
CH3OH (1 : 1, v/v, 5 mL) and pre-purified by automated flash-
column chromatography over reversed-phase C18 silica gel (system
I). The product-containing fractions were lyophilised and the
resulting amorphous powder was purified twice by semi-
preparative RP-HPLC (system E, tR = 41.0-51.0 min and system F, tR =
34.6-37.2 min). The TFA salt of desired pyronin 6 was recovered as
red amorphous powder (6 mg, 2%). Please note: despite two RP-
HPLC purifications, the product remains contaminated with a minor
12.2 (2 C, CH3-Et), 43.3, 44.1 (2 C, CH2-Et), 100.6, 107.7, 108.2,
112.5, 113.8, 115.2, 126.5, 128.2 (2 C), 129.0 (2 C), 130.0, 130.1,
135.8, 140.7, 153.4, 157.6, 160.4, 169.2, 185.1 ppm; HPLC (system
A): tR = 5.2 min (purity 99% at 260 nm); LRMS (ESI+, recorded during
+
RP-HPLC analysis): m/z 403.1 [M + H]+ (100), calcd for C25H27N2O3
403.2; LRMS (ESI-, recorded during RP-HPLC analysis): m/z 400.8 [M
- H]- (100) and 447.0 [M - H + FA]- (60), calcd for C25H25N2O3- 401.2;
HRMS (ESI+): m/z 425.18194 [M + Na]+, calcd for C25H26N2O3Na+
425.18356; UV-vis: λmax(PBS)/nm 264 and 368 (ε/dm3 mol-1 cm-1 14
860 and 16 040).
amount of starting aldehyde (10%). IR (ATR):
ν = 3334 (broad), 3109
LAP-sensitive probe (5). A mixture of benzaldehyde 1 (76 mg, 0.39 (broad), 2923, 2855, 2740, 1650 (broad), 1572 (broad), 1488, 1429,
mmol, 1.7 equiv.), iodoaryl derivative 3 (100 mg, 0.23 mmol, 1 1381, 1336, 1274, 1149, 1118, 1072, 1008, 967, 822, 796, 737, 703
equiv.), finely ground K3PO4 (93 mg, 0.44 mmol, 1.9 equiv.), CuI (5 cm-1; 1H NMR (500 MHz, CD3OD):
δ = 1.32 (t, J = 7.0 Hz, 6 H, CH3-Et),
mg, 0.02 mmol, 0.1 equiv.) and picolinic acid (6 mg, 0.04 mmol, 0.2 3.70 (q, J = 7.0 Hz, 4 H, CH2-Et), 6.77 (bd, J = 1.0 Hz, 1 H, H-Ar), 6.92
equiv.) in dry DMSO (1 mL) was heated in a sealed tube at 90 °C (bd, J = 2.0 Hz, 1 H, H-Ar), 6.95 (dd, J = 2.0 Hz, J = 9.0 Hz, 1 H, H-Ar),
overnight. The reaction was checked for completion by HPLC 7.18 (dd, J = 2.0 Hz, J = 9.0 Hz, 1 H, H-Ar), 7.72 (d, J = 9.0 Hz, 1 H, H-
(system A) and quenched with 10 mL of deionised water. Then, the Ar), 7.80 (d, J = 9.0 Hz, 1 H, H-Ar), 8.55 (s, 1 H, H-Ar) ppm; 13C NMR
resulting mixture was extracted with DCM, washed with aq. 1 N HCl, (126 MHz, CD3OD):
dried over anhydrous Na2SO4, filtered and concentrated under 98.6, 115.5, 115.7, 116.0, 118.0, 134.8, 135.2, 147.6, 157.9, 159.8,
reduced pressure. The residue was retaken in DCM (400 µL) and 160.3, 162.1 ppm; 19F NMR (470 MHz, CD3OD):
= -76.9 (s, 3 F, CF3-
δ = 12.9 (2 C, CH3-Et), 47.0 (2 C, CH2-Et), 97.3,
δ
TFA (700 µL, 40 equiv.) was added at 0 °C. The resulting reaction TFA); HPLC (system B): tR = 3.7 min (purity 92% at 260 nm and 99%
mixture was stirred at 0 °C for 20 min, diluted in CH3CN (5 mL) and at 500 nm); LRMS (ESI+, recorded during RP-HPLC analysis): m/z
evaporated under vacuum. The resulting oily residue was purified 267.0 [M + H]+ (100), calcd for C17H19N2O+ 267.1; Elemental anal.:
by semi-preparative RP-HPLC (system D, tR = 18.0-22.0 min). The Found C, 56.6; H, 5.7; N, 6.8. C17H18N2O . 1.33 CF3CO2H requires C,
TFA salt of desired LAP-sensitive probe 5 was recovered as white 56.5; H, 4.7; N, 6.7%. UV-vis: λmax(PBS)/nm 527 (ε/dm3 mol-1 cm-1 70
powder (13 mg, yield 11%). IR (ATR):
1485, 1441, 1393, 1355, 1267, 1199, 1135, 1100, 977, 834, 798, 721
cm-1; 1H NMR (300 MHz, DMSO-d6):
= 0.90 (d, J = 3.6 Hz, 6 H, CH3-
ν = 2970, 1669, 1591, 1524, 350); Fluorescence: λmax(PBS)/nm 544 (ΦF 7%).
δ
Acknowledgements
Leu), 1.06 (t, J = 7.2 Hz, 6 H, CH3-Et), 1.63 (bd, 3 H, CH-CH2-Leu),
3.37 (mask by H2O signal) (q, J = 6.9 Hz, 4 H, CH2-Et), 3.88 (bs, 1 H,
CH-NH2-Leu), 6.16 (d, J = 2.4 Hz, 1 H, H-Ar), 6.64 (dd, J = 2.1 Hz, J =
9.0 Hz, 1 H, H-Ar), 6.74 (m, 1 H, H-Ar), 7.34-7.40 (m, 3 H, H-Ar), 7.67
(d, J = 9.0 Hz, 1 H, H-Ar), 8.24 (bs, 2 H, NH2-Leu), 9.87 (s, 1 H, H-
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., are
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
formyl), 10.60 (s, 1 H, NH) ppm; 13C NMR (126 MHz, DMSO-d6):
δ =
12.2 (2 C, CH3-Et), 21.8, 22.6, 23.7, 39.5 (1C, masked by DMSO
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Organic & Biomolecular Chemistry
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8 | Org. Biomol. Chem., 2017, 00, 1-3
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ARTICLE
+
Et2N
O
O
NEt2
Et2N
O
O
NEt2
+
PO(OEt2)Cl
(Sarin mimic)
Cl-
Et2N
O
NEt2
Cl-
C6H5Cl
- PO(OEt2)OH
EtO
EtO
P
pyronin B
NA570
Lei and Yang 2014
O
Et2N
O
NEt2
Et2N
O
NEt2
+
Et2N
O
NEt2
S
S
Hg(II)
X-
S
S
HEPES buffer solution
pH 7.4 (5% DMSO)
- H+, -
Hg
Hg570
Song et al. 2014
pyronin B
S
+HgS
H
R
N
O
NEt2
H2N
O
NEt2
HN
O
NEt2
protease
O
- H2O
phosphate buffer solution
pH 7.5
O
O
unsymmetrical pyronin
This work
Fig. 1 Background information about the "covalent-assembly" principle applied to in vitro fluorogenic detection of organophosphorus nerve agents
(Sarin mimics) and Hg(II) cation through in situ synthesis of pyronin B (X- = anionic form of HEPES), and its extension to protease sensing explored in
this work (unsymmetrical pyronin = 6-N,N-diethylamino-3H-xanthen-3-imine = compound
6
).
O
POCl3 , DMF
H
75 °C, 2 h
then
RT, overnight
Et2N
OH
Et2N
OH
1
yield 83%
O
H
Et2N
O
O
N
CuI, K3PO4,
TEA, PhAcCl
O
N
CO2H
H
+
O
THF, 0 °C to RT, 2 h 30
DMSO, 130 °C, overnight
I
N
I
NH2
Et2N
OH
H
yield 90%
yield 54%
1
4
2
O
1. CuI, K3PO4,
O
1. IBCF, NMM, THF
O
N
CO2H
H
NHBoc
-20 °C to -10 °C, 25 min
Et2N
O
N
DMSO, 90 °C, overnight
H
NHBoc
HO
NH3
O
I
N
+
H
O
Et2N
OH
2. TFA, DCM, 0 °C, 20 min
3
O
2.
1
yield 11%
(after RP-HPLC purification)
O
I
NH2
Boc-L-Leu-OH
5
F3C
THF, -10 °C to 5 °C , 3 h
yield 70%
Scheme 1 Synthesis of protease-sensitive fluorescence "turn-on" probes 4 and 5 based on the "covalent-assembly" principle (Boc = tert-butyloxycarbonyl,
IBCF = isobutyl chloroformate, NMM = N-methylmorpholine, PhACl = phenylacetyl chloride).
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DOI: 10.1039/C7OB00370F
ARTICLE
O
O
O
O
O
LAP, aq. buffer, 37 °C
PGA, aq. buffer, 37 °C
O
NH2
Ph
Et2N
O
N
Et2N
N
fast
H-L-Leu-OH
fast
H
H
Et2N
O
NH2
4
5
PhAcOH
aniline-intermediate characterized
by HPLC-MS analysis
acid cat. in
acetate buffer
(pH 5.6)
H
H
R
O
HN
O
H2N
R
R-NH2 = NH3 ((NH4)2SO4 as source), H-Leu-OH,
aniline intermediate or lysine residues of enzyme
Et2N
O
NH2
Et2N
NH2
- H2O
imine formation by
reaction with R-NH2
?
R
R
NH
NH2
O
H
Abs/Em = 527/544 nm
QY = 7% in PBS
- R-NH2
Et2N
O
NH2
Et2N
O
NH2
Et2N
NH2
unsymmetrical pyronin 6
OH
O
if cyclisation-aromatisation domino process does
not involve imine formation, pyronin 6 will come
from dehydration of xanthydrol derivative (ROH)
Et2N
NH2
Fig. 4 Proposed detection mechanism of proteases via the "covalent-assembly" principle, supported by fluorescence-based in vitro assays and HPLC-
fluorescence/-MS analyses performed with PGA-sensitive probe and LAP-sensitive probe
4
5.
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