Fluorogenic Behaviour of the Hetero-Diels–Alder Ligation of 5-Alkoxyoxazoles with Maleimides and their Applications

Article abstract of DOI:10.1002/chem.201603617

Fluorogenic reactions are largely underrepresented in the toolbox of chemoselective ligations despite their tremendous potential, particularly in chemical biology and biochemistry. In this respect, we have investigated in full detail the fluorescence behaviour of the azaphthalamide, a scaffold which is generated through a hetero-Diels–Alder reaction of 5-alkoxyoxazole and maleimide derivatives under mild conditions that are compatible with, among others, peptide chemistry. The scope and limitations of such a fluorogenic labelling strategy were examined through four distinct applications, which target enzymatic activities or bioorthogonal reactions.

Full text of DOI:10.1002/chem.201603617

DOI: 10.1002/chem.201603617
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Fluorescence Probes
Fluorogenic Behaviour of the Hetero-Diels–Alder Ligation of
5-Alkoxyoxazoles with Maleimides and their Applications
Kꢀvin Renault,[a] Laurie-Anne Jouanno,[b] Antoine Lizzul-Jurse,[a] Pierre-Yves Renard,[a] and
Cyrille Sabot*[a]
Abstract: Fluorogenic reactions are largely underrepresent-
ed in the toolbox of chemoselective ligations despite their
tremendous potential, particularly in chemical biology and
biochemistry. In this respect, we have investigated in full
detail the fluorescence behaviour of the azaphthalamide,
a scaffold which is generated through a hetero-Diels–Alder
reaction of 5-alkoxyoxazole and maleimide derivatives under
mild conditions that are compatible with, among others,
peptide chemistry. The scope and limitations of such a fluo-
rogenic labelling strategy were examined through four dis-
tinct applications, which target enzymatic activities or bioor-
thogonal reactions.
alkenes[9] and between o-quinone methides and vinyl thioeth-
ers.[10]
Introduction
Chemoselective ligation chemistry has dramatically gained im-
portance in recent years, which provides fascinating opportuni-
ties in different research fields, including material and life sci-
ences.[1] It involves reliable covalent bond formation, which
takes place specifically between two chemical handles under
mild conditions and avoids significant interference with biolog-
ical functional groups. This strategy enables effective construc-
tion of (bio)molecular structures that span from simple scaf-
folds to complex molecular architectures, which is enabled by,
among other characteristics, their broad functional group toler-
ance and reaction condition tolerability.[2]
Specifically, some ligation strategies have been used to
design molecular biosensors and bioprobes for real-time moni-
toring of biological events or enzymatic assays suitable for
high-throughput screening or biomedical imaging applica-
tions.[11] The preparation of such biomolecular systems mostly
relies on a fluorescent labelling step, which involves chemose-
lective ligation tools. In most cases, a fluorescent dye is re-
quired on the ligation partner to label the molecule of interest;
however, there are a few scarce examples of fluorogenic reac-
tions[12] that enable the formation of fluorescent linkages, such
as the aldehyde–amino benzamidoxime ligation,[13] the tetra-
zole–alkene photoclick chemistry,[14] or azide–cyclooctyne liga-
tion with specifically designed cyclooctynes.[15]
Different ligation strategies have been proposed over the
years, and condensation reactions of hydrazines/alkoxyamines
with carbonyl derivatives to provide the corresponding hydra-
zones/oximes were among the first chemoselective ligations to
be reported.[3] Azides have proven to be a particularly effective
reaction partner, first with triarylphosphines in the Staudinger
ligation,[4] and then with alkynes in the copper-catalyzed
azide–alkyne cycloaddition (CuAAC).[5] Later on, strain-promot-
ed azide–alkyne (SPAA),[6] alkyne–sydnone,[7] and alkyne–cyclic
nitrone cycloaddition (SPANC)[8] were developed to circumvent
the use of toxic copper catalysts. Several other ligation strat-
egies have been proposed, which include inverse-electron-
demand Diels–Alder reactions between tetrazines and strained
We recently reported the Kondrat’eva ligation, an irreversi-
ble-based Diels–Alder reaction between 5-alkoxyoxazole and
maleimide derivatives, which led to the formation of an azaph-
thalimide linker.[16,17] Interestingly, this irreversible two-step re-
action, which involves a Diels–Alder reaction followed by an ar-
omatization step with concomitant release of ethanol, oc-
curred in a single-step protocol either in the presence of a few
[a] K. Renault, A. Lizzul-Jurse, Prof. Dr. P.-Y. Renard, Dr. C. Sabot
Normandie University, CNRS, UNIROUEN, INSA Rouen
COBRA (UMR 6014), 76000 Rouen (France)
E-mail: cyrille.sabot@univ-rouen.fr
[b] Dr. L.-A. Jouanno
Department of Chemistry and Biomolecular Sciences
University of Ottawa, 10 Marie Curie, Ottawa, Ontario, K1N 6N5 (Canada)
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under:
http://dx.doi.org/10.1002/chem.201603617.
Scheme 1. Phthalimide and azaphthalimide derivatives.
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equivalents of trifluoroacetic acid (TFA) in the medium or in
a sodium acetate buffer (pH 5.0). Shortly after publishing our
article, Song et al. reported a phthalimide-based fluorescent
probe for the detection of biothiols (Scheme 1a).[18] Their result
led us to examine whether the azaphthalimide scaffold that is
generated during the ligation process would be fluorescent,
and if so, to what extent (Scheme 1b). Herein, we report the
detailed investigation of the photophysical properties of
azaphthalimide scaffolds along with their direct applications in
the synthesis of biologically relevant fluorogenic probes.
charge transfer (ITC) process, which is likely responsible for
fluorescence emission. Consequently, the absorption and emis-
sion behaviour of the azaphthalimide scaffold was investigated
in aqueous media at different pH values to determine the suit-
ability of the azaphthalimide scaffold for future fluorescence
applications.
Results and Discussion
Study of photophysical properties of azaphthalimides
The optical behaviour of the azaphthalimide scaffold (i.e., sol-
vent and pH effect) was determined by using the model com-
pound 1. First, absorption and emission spectra were recorded
in various solvents, which ranged from nonpolar aprotic to
polar protic medium (Figure 1a).
In weakly ionizing solvents, such as toluene or dichlorome-
thane, an absorption band at ꢀ350 nm was mainly observed.
In contrast, in polar aprotic and polar protic solvents, such as
DMF, MeOH, or water, a new absorption peak was detected
above 400 nm along with a decrease in the intensity of the
band at ꢀ350 nm. From these positive solvatochromic results,
it was assumed that the high- and low-energy absorption
band could be assigned to neutral and ionized azaphthalimide
species, respectively. In fact, polar media would favor the for-
mation of a hydrogen-bonding interaction between the hy-
droxyl group of the azaphthalimide moiety and the solvent. To
further investigate this hypothesis, the influence of the depro-
tonation of the hydroxyl group of azaphthalimide 1 on the ab-
sorption and fluorescence emission spectra was investigated in
the nonpolar toluene solvent (Figure 1b–d). The absorption
peak at ꢀ350 nm decreased gradually upon the addition of
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and the appearance
of a red-shifted absorption band was observed at ꢀ420 nm.
Moreover, the gradual disappearance of the fluorescence emis-
sion that corresponded to an excitation in the high-energy ab-
sorption band (350 nm) was observed, and
a band at
ꢀ520 nm appeared. Besides, a noticeable increase in the emis-
sion peak intensity at ꢀ520 nm under an excitation wave-
length of 430 nm was observed. Collectively, these data sug-
gest that the neutral azaphthalamide species absorbs at
350 nm and emits at 420 nm, and the phenolate species ab-
sorbs at 420 nm and emits at 520 nm. These results are also
confirmed by the analysis of their excitation spectra recorded
at 420 and 520 nm (see the Supporting Information). In most
of the aforementioned solvents (except toluene and dichloro-
methane), both phenol and phenolate species are present in
the reaction medium, albeit in different proportions. Further-
more, the phenolic form of the azaphthalimide generally ex-
hibits a significantly weaker fluorescence at 420 nm than its
phenolate counterpart at 520 nm. Indeed, deprotonation of
the hydroxyl group increases the electron density within the
aromatic ring system and then enhances the intramolecular
Figure 1. Photophysical behaviour of compound 1. a) Absorption spectra in
different solvents and b) in toluene in the presence of different amounts of
DBU at 258C. Emission spectra in toluene in the presence of different
amount of DBU upon an excitation at c) 350 nm and d) 430 nm at 258C.
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The spectroscopic data of compound 1 were recorded in
aqueous solutions of pH 1.1–10.9 (Figure 2). A decrease in pH
value resulted in a decrease in intensity of the absorption
band at ꢀ420 nm and a concomitant increase in intensity of
the absorption band at ꢀ350 nm, with an isosbestic point at
378 nm. Next, the fluorescence spectra were determined at an
excitation wavelength of 430 nm for different pH values. As ex-
pected from the absorption spectra, the emission intensity de-
creased as the pH of the solution was lowered. From this
study, it appears that the phenolate ion (i.e., the most fluores-
cent species) is the predominant form at pH values higher
than 6.0, which corresponds to the pKa value that was calculat-
ed from the absorbance values at 420 nm at different pH
values (see the Supporting Information). To quantify this trend
more precisely, the fluorescence quantum yield of compound
1 was measured at different pH values (pH 2.2–10.9, Table 1).
Under an excitation wavelength of 430 nm, acceptable quan-
tum yields were observed for pH values higher than 5.6. Fol-
lowing these encouraging results, the photophysical properties
of a series of diversely functionalized azaphthalimides were
subsequently determined to assess the impact of substituents
on the photoluminescence characteristics of the azaphthali-
mide and thus understand specific requirements for optimal
fluorescence emission (entries 10–13). 2-Benzyl-subsituted pyri-
dine systems exhibited quantum yields at pH 7.4 that were
comparable to that of compound 1 (0.07–0.10, entries 11–13)
with the exception of unsubstituted compound 2, which dis-
Figure 2. a) Absorption and b) emission spectra of compound 1 at 258C
upon an excitation at 430 nm at different pH values.
Table 1. Spectroscopic behaviour of azaphthalimides 1–8.
[b]
Entry
Cmpd
R1
R2
R3
pH
labs [nm][a]
lem [nm][a]
e [mꢁ1 cmꢁ1
]
FF
1
2
3
4
5
6
7
8
1
Bn
Me
Me
2.2
4.5
5.6
6.7
7.0
7.4
8.5
9.9
10.9
7.4
7.4
7.4
7.4
2.2
4.5
5.6
6.7
7.0
7.4
8.5
9.9
10.9
7.4
7.4
420
420
420
417
418
417
417
418
417
407
418
421
418
415
417
417
415
413
416
415
415
415
416
418
522
522
522
522
520
520
520
520
521
513
521
521
519
520
518
519
517
521
517
520
518
517
518
519
3300
6300
9400
12900
14000
13700
11600
12000
<0.01
0.01
0.04
0.08
0.09
0.09
0.11
0.09
0.09
0.02
0.10
0.07
0.10
0.08
0.05
0.07
0.14
0.15
0.15
0.17
0.16
0.16
0.16
0.18
9
10700
[c]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2
3
4
5
6
Bn
Bn
Bn
Bn
H
Me
Me
Ph
(CH2)5CO2H
Me
[c]
[c]
[c]
CH2Bn
Me
Me
(CH2)5CO2H
Me
1300
5000
4700
5900
6000
6500
6000
6400
7500
[c]
7
8
(CH2)5CONHCHPh(Et)
(CH2)5CONHCHPh(Et)
Me
Me
Me
[c]
(CH2)5CONHBn
[a] labs is the maximum of the absorption band responsible for the emission observed at lem. [b] Measurement determined at 258C by a relative method
that used Lucifer yellow as standard (FF =0.21 in water) with excitation at 430 nm. [c] Not determined.
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played a markedly lower quantum yield (0.02, entry 10). Ac-
cordingly, the presence of an aliphatic group at position 6 of
the pyridine ring system is crucial for fluorescence enhance-
ment. Satisfyingly, conjugatable or conjugated hexanoic-acid-
based derivatives 6–8 exhibited appreciable quantum yields in
the range of 0.15–0.18, which is even higher than those of
their 2-benzyl-substituted congeners, but with lower levels of
brightness.
Applications of the fluorogenic Kondrat’eva ligation
The scope and limitations of such a fluorogenic labelling strat-
egy were examined through four distinct applications, which
are detailed hereafter.
Peptide labelling (Application 1)
The chemical modification of proteins or peptides that are
only available in small quantities is generally conducted with
an excess of more readily accessible labelling agents to com-
pensate for the dilution conditions. In this context, a fluorogen-
ic ligation reaction would advantageously avoid unwanted
fluorescence emission from the unreacted starting material,
which was used in excess and may not be easily removed from
the product. To illustrate this point, the heptapeptide 9, pre-
pared through standard solid-phase synthesis, was functional-
ized with the maleimide 10. The corresponding maleimide-
modified heptapeptide 11 was treated with a three molar
excess of oxazole 12 in NMP in the presence of 1 equivalent of
TFA (Scheme 2). It is worth noting that, when in aqueous
media, the Kondrat’eva ligation could proceed even in the
presence of a large excess of TFA owing to the buffering effect
of water; however, in organic media, TFA should not exceed
one or two equivalents relative to the oxazole (see the Sup-
porting Information). The resulting RP-HPLC analysis with UV
detection of the reaction mixture clearly showed the presence
of two close peaks at 9.58 and 9.63 min, which correspond to
the excess of starting oxazole 12 and the azaphthalimide prod-
uct 13, respectively, and were inseparable by semi-preparative
RP-HPLC (Figure 3). Advantageously, in the fluorescence detec-
Figure 3. HPLC chromatogram of the ligation step that involves the starting
materials 11 and 12 and the product 13, in the a) UV and b) fluorescence
(lex =420 nm, lem =520 nm) detection mode.
tion mode, only the peak that corresponded to the azaphthali-
mide conjugate 13 was observed on the chromatogram (along
with the peptide product in a different protonation state at
10.29 min), the unreacted oxazole peak being no longer visi-
ble.
Synthesis of a coumarin–azaphthalimide-based FRET cassette
(Application 2)
Fluorescent or fluorogenic molecular systems have become
very popular chemical tools in diverse fields of research, such
as in medicinal chemistry for the determination of enzymatic
activity or in chemical biology for real-time monitoring and
Scheme 2. Synthetic route to fluorescent peptide 13 from peptide 11 and oxazole 12.
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imaging of biological events. In this respect, the spectrum of
use of azaphthalimide dyes for fluorescence applications was
explored through the design of three different FRET-based
probes. Such molecular systems involve a donor fluorophore,
which transfers its excitation energy to a nearby acceptor chro-
mophore (fluorophore or quencher) in a nonradiative manner.
To optimize the FRET process, the donor emission spectrum
should significantly overlap the acceptor absorption spectrum.
Initially, the construction of a FRET cassette was investigated
through the covalent attachment of two fluorophores that dis-
played substantial spectral overlap (see the Supporting Infor-
mation): a coumarin dye 14 as the donor, recently reported by
us,[19] and an azaphthalimide scaffold 6 as the acceptor
(Scheme 3). Complete photophysical properties of the corre-
sponding FRET pair 16, which was obtained in 90% yield from
compound 15, were determined to establish whether the
spectral properties of each fluorophore were preserved upon
their conjugation. The UV/Vis absorption spectrum of the FRET
cassette 16 showed two absorption maxima at 325 and
420 nm, which is in accordance with the sum of absorption
bands of the parent dyes (Figure 4).
Synthesis of an azaphthalimide–tetrazine-based smart probe
(Application 3)
The preparation of a bioorthogonal smart probe based on
a novel fluorophore/quencher FRET pair was envisioned. In this
approach, the enhancement in fluorescence emission intensity
was selectively triggered through a bioorthogonal process. Ac-
cordingly, this recent labelling strategy enabled direct fluores-
cence imaging of diverse biological targets without the remov-
al of excess unreacted probe by several washing steps. Al-
though significant progress has been made in the develop-
ment of tetrazine-based smart probes by using BODIPY, cou-
marin, or fluorescein fluorophores, we seized the opportunity
to increase their limited number, specifically by using a readily
accessible fluorogenic probe.
Tetrazine 17 displays an absorption maximum of ꢀ521 nm,
which also corresponds to the emission maximum of the
azaphthalimide ring system. Consequently, a tetrazine–azaph-
thalimide-based FRET probe was envisioned and achieved
through a final amide bond formation step between the
amino tetrazine 17 and the carboxylic acid moiety of the
azaphthalimide 18 (Scheme 4). With the probe in hand, its flu-
orogenic behaviour was explored through the inverse-elec-
tron-demand Diels–Alder cycloaddition with the strained
alkene norbornene 20 in phosphate-buffered saline (PBS,
pH 7.4) at room temperature. Satisfyingly, a significant four-
fold fluorescence turn-on response was observed at 520 nm
within 10 min of the reaction commencement (Figure 5). This
result demonstrates that the fluorescence of the azaphthali-
mide scaffold could be quenched by the tetrazine core, pre-
sumably through a FRET mechanism. Thus, reaction of the tet-
razine moiety with the strained alkene could be directly fol-
lowed in real time through the fluorescence recovery of the
As expected from the FRET strategy, the azaphthalimide dye
could now be selectively excited at 315 nm, while allowing
emission in the green region of the spectrum (Figure 4c,
dotted curve). Thus, a large pseudo-Stokes shift of 195 nm was
observed, which advantageously avoids any self-absorption
phenomenon that standard fluorophores may suffer from.
Moreover, the energy-transfer efficiency (ETE) was found to be
77%, which demonstrates the successful combination of these
two fluorophores, although significant residual fluorescence at-
tributed to the coumarin moiety was still observed at 325 nm.
This latter result highlights the difference in fluorescence effi-
ciency between those two fluorophores.
Scheme 3. Preparation of the coumarin–azaphthalimide-based FRET cassette 16.
Figure 4. Normalized absorption (dashed line), emission (dotted line, lex =315 nm for compounds 14 (a), 16 (c) and lex =430 nm for compound 6 (b)), and ex-
citation spectra (black line, lem =395 nm for compounds 14 (a), 16 (c); grey line, lem =520 nm for compounds 6 (b), and 16 (c)) in PBS pH 7.4 at 258C.
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Scheme 4. Preparation of the azaphthalimide–tetrazine conjugate 19.
Figure 5. a) Fluorescence emission spectrum (“Scan” mode, lex =420 nm) of probe 19 before (black) and after (grey) addition of a 3-fold excess of norbornene.
b) Fluorogenic activation time course (“kinetic” mode, lex =420 nm, lem =520 nm) of probe 19 in PBS pH 7.4 at 258C.
azaphtalimide moiety. Notably, probe 19 could advantageously
be used to compare the kinetics of tetrazine-based Diels–Alder
reactions with various dienophiles.[20]
which issued a satisfying ten-fold increase in fluorescence at
520 nm after enzymatic digestion (Figure 6).
Conclusion
Synthesis of a fluorogenic substrate of urokinase (Application
4)
This study reports the detailed photophysical properties of the
azaphthalimide, a green-emitting moiety that was generated
through the ligation of the nonfluorescent oxazole and malei-
mide moieties. This work demonstrated that azaphthalimides
display solvatochromic character owing to hydrogen-bonding
interactions with the solvent, for which the most fluorescent
species (i.e., phenolate) is favored in polar or basic media as
well as in physiological conditions. Finally, the albeit moderate
fluorescence behaviour of azaphthalimides has found useful
applications, in particular for the construction of fluorogenic
FRET-based probes to target specific enzymatic activities or to
evaluate the efficacy of bioconjugate processes, such as the
tetrazine ligation. The latter application is currently being in-
vestigated in our group.
The final investigation relied upon the preparation of a FRET-
based urokinase-plasminogen-activator(uPA)-sensitive probe;
uPA is a diagnostically relevant serine protease owing to its
overexpression in breast and prostate cancer. Accordingly, the
fluorogenic probe 24, which constituted a FRET pair of an
azaphthalimide fluorophore and a newly prepared dabcyl-like
quencher 21[21] that were connected at both ends of octapep-
tide HꢁSerꢁGlyꢁArgꢁSerꢁAlaꢁAsnꢁAlaꢁLysꢁNH2 (known uPA
substrate),[16,22] was prepared in three steps from compound 9.
During the first two steps, the diazo quencher 21 and the mal-
eimide 10 were consecutively attached at the N- and C-termi-
nal of the octapeptide, respectively. Then, the hetero-Diels–
Alder reaction of intermediate 23 with the oxazole 12 afforded
the corresponding conjugate in 38% yield. The structure of
the resulting probe was unambiguously confirmed by HRMS
analysis (Scheme 5).
Experimental Section
Results obtained from the proteolysis of the fluorogenic
probe 24 with commercial uPA are summarized in Figure 6.
Even though an important quenching of fluorescence was ob-
served before the enzymatic digestion, the fluorescence was
completely recovered in the presence of uPA within 30 min.
The quenching efficiency (QE) was determined to be 93%,
General information
Flash column chromatography purifications were performed on
silica gel (40–63 mm). Thin-layer chromatography (TLC) were car-
ried out on Merck DC Kieselgel 60 F-254 aluminum sheets. The
spots were visualized by illumination with a UV lamp (at 254/
365 nm) and/or staining with KMnO4 solution. Unless otherwise
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Scheme 5. Synthesis of probe 24.
Figure 6. a) Fluorescence emission spectrum (“Scan” mode, lex =420 nm) of probe 24 before (black) and after (grey) incubation with uPA in PBS pH 7.4 at
378C. b) Fluorogenic activation time course (“kinetic” mode, lex =420 nm, lem =520 nm) of probe 24 in PBS pH 7.4 at 378C.
noted, all chemicals were used as received from commercial sour-
ces without further purification. PBS (pH 7.4, 0.1m), carbonate
buffer (pH 10.89, 0.1m), and aq. mobile phases for HPLC were pre-
pared with water that was purified by means of a MilliQ system
(purified to 18.2 MWcm).
formed by using an ultra-micro quartz cell (Hellma, 105.251-QS,
20ꢂ3 mm, chamber volume=45 mL). Excitation/emission spectra
were recorded under the same conditions after emission/excitation
at the corresponding wavelength (520/430 nm, excitation and
emission filters: auto, excitation and emission slit=5 nm). Fluores-
cence quantum yields were measured at 258C by a relative
method that used Lucifer yellow (FF =21% in water, 430 nm) as
a standard.[23] The following Equation (1) was used to determine
the relative fluorescence quantum yield in which A is the absorb-
ance (in the range of 0.01–0.1 a.u.), F is the area under the emis-
sion curve, n is the refractive index of the solvents (at 258C) used
in measurements, and the subscripts s and x represent standard
and unknown, respectively.
Instrument and methods
1H and 13C NMR spectra were recorded on a 300 MHz spectrometer.
Chemical shifts are expressed in parts per million [ppm] by using
the residual solvent peak for calibration. J values are expressed in
Hz. High resolution mass spectra (HRMS) were obtained by using
an orthogonal acceleration time-of-flight (oa-TOF) mass spectrome-
ter equipped with an electrospray source in positive and negative
modes. Low resolution mass spectra (LRMS) were obtained with
a mass spectrometer equipped with an ESI source. UV/Vis absorp-
tion spectra were obtained by using a rectangular quartz cell (stan-
dard cell, Open Top, 10ꢂ10 mm, chamber volume=3.5 mL). Fluo-
rescence spectroscopic studies (emission/excitation spectra) were
performed by using a semi-micro quartz fluorescence cell (Hellma,
104F-QS, 10ꢂ4 mm, chamber volume=1.4 mL for quantum yields
determination, chamber volume=3.5 mL for all other studies). In
vitro fluorescence assays and in vitro enzymatic assays were per-
2
ð1Þ
FFðXÞ ¼ ðAs=AxÞ ðFx=FsÞ ðnx=nsÞ FFðSÞ
The following refractive index values were used: 1.337 for PBS,
1.333 for water, 1.362 for EtOH, 1.497 for toluene, 1.424 for CH2Cl2,
1.344 for MeCN, 1.407 for THF, 1.328 for MeOH, 1.431 for DMF,
and 1.478 for DMSO.
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wavelength used for the detection was performed at 340 and
240 nm.
Preparation of the carbonate buffer (pH 10.89, 0.1m)
500 mL of a 0.1 m solution of NaHCO3 and 500 mL of a 0.1m solu-
tion of Na2CO3·10H2O were prepared. 0.1m solution of NaHCO3
(50 mL) was added to the 0.1m solution of Na2CO3·10H2O (450 mL)
to give, after the pH value was adjusted by using a pH-meter, the
carbonate buffer (pH 10.89, 0.1m).
Synthesis of azaphthalimide peptide conjugate 13
Step 1: a) N-Hydroxysuccinimide (211 mg, 1.83 mmol, 1.1 equiv)
and EDCI·HCl (351 mg, 1.83 mmol, 1.1 equiv) were added to a solu-
tion of maleimide carboxylic acid 10[24] (355 mg, 1.67 mmol,
1 equiv) in a mixture of dry THF/EtOH (6 mL, 1:1 v/v). The resulting
reaction mixture was stirred at room temperature until reaction
completion (cycloexane/EtOAc 50:50). The solution was quenched
with a saturated solution of NaHCO3, extract with CH2Cl2 (2ꢂ
20 mL). The combined organic phases were washed with brine,
dried over MgSO4, concentrated under vacuum, and used in the
next step without further purification. b) The TFA salt of the hepta-
peptide 9[16] (20 mg, 18.7 mmol, 1 equiv) and N-hydroxysuccini-
mide-activated maleimide 10 (7 mg, 22.5 mmol, 1.2 equiv) were dis-
solved in NMP (330 mL). DIEA (2.0m solution in NMP, 26.1 mL,
0.15 mmol, 8 equiv) was added, and the resulting reaction mixture
was stirred at RT for 2 h. The reaction progress was monitored by
RP-HPLC (System QC). After the reaction was completed, Et2O was
added, and the resulting precipitate was centrifuged. The precipi-
tate that contained the maleimide-modified peptide was collected
and used without further purification in the next step, because we
found that the conjugate was not stable during purification on
semi-preparative RP-HPLC
Step 2: 5-Ethoxyoxazole 12[16] (18 mg, 74.8 mmol, 4 equiv) and a so-
lution of TFA (1.43 mL, 18.7 mmol, 1 equiv) in water (210 mL) was
added to the peptide–maleimide conjugate, which was obtained
in Step 1, dissolved in DMSO (420 mL), and the reaction mixture
was stirred at RT for 5 h. The reaction progress was monitored by
RP-HPLC (System QC). The mixture was diluted with aq. 0.1% TFA
and purified by semi-preparative RP-HPLC, and the product-con-
taining fractions were lyophilized (System F). To remove the excess
oxazole, which was not separable from the azaphthalimide product
by semi-preparative RP-HPLC, Et2O was added to the lyophilisate,
the resulting precipitate was centrifuged, and the product-contain-
ing fractions were lyophilized to give 10.5 mg (42% yield for the
two steps) of the azaphthalimide-labelled peptide 13 as a yellow
amorphous powder. UV/Vis (PBS): lmax =417 nm; HPLC (Sys-
tem QC): tR =21.883 min (93% purity).
HPLC systems used for purity control or purification
Analytical HPLC
System QC1: Accucore C18 column (2.1ꢂ150 mm) with MeCN am-
monium acetate buffer (20 mm, pH 7.0) as a linear gradient over
35 min from 10–90% of MeCN at a flow rate of 0.4 mLminꢁ1 and
injection volume of 1 mL. UV/Vis wavelength used for the absorb-
ance detection was performed at 230 nm, and UV/Vis wavelength
used for the fluorescent detection was performed at 420 nm for
excitation and 520 nm for emission.
System QC: Thermo Hypersyl GOLD C18 column (5 mm, 2.1ꢂ
100 mm) with MeCN and 0.1% aq. TFA as eluents (0% MeCN for
5 min followed by 0–100% MeCN for 40 min) at a flow rate of
0.25 mLminꢁ1
.
Semi-preparative HPLC
Several chromatographic systems were used for the purification
steps:
System A: RP-HPLC (Varian Kromasil C18 column, 10 mm, 10.0ꢂ
250 mm) with MeCN and 0.1% aq. TFA as eluents (0% MeCN for
5 min, 0–30% MeCN for 30 min, an isocratic mixture of 30% MeCN
for 15 min, and 30–100% of MeCN for 55 min) at a flow rate of
7 mLminꢁ1. UV/Vis wavelength used for the detection was per-
formed at 465 nm.
System B: RP-HPLC (Varian Kromasil C18 column, 10 mm, 21.2ꢂ
250 mm) with MeCN and 0.1% aq. TFA as eluents (0% MeCN for
5 min, 0–100% MeCN for 100 min, and 100% MeCN for 45 min) at
a flow rate of 20 mLminꢁ1. UV/Vis wavelength used for the detec-
tion was performed at 570 and 430 nm.
System C: RP-HPLC (Thermo Hypersil GOLD C18 column, 5 mm,
10.0ꢂ250 mm) with MeCN and 0.1% aq. TFA as eluents (0% MeCN
for 10 min, 0–100% MeCN for 200 min, and 100% MeCN for
30 min) at a flow rate of 4 mLminꢁ1. UV/Vis wavelength used for
the detection was performed at 575 and 390 nm.
Synthesis of the coumarin FRET pair 16
Compounds 14 and 15 were prepared according to a reported
System D: RP-HPLC (Varian Kromasil C18 column, 10 mm, 21.2ꢂ
250 mm) with MeCN and 0.1% aq. TFA as eluents (0% MeCN for
10 min, 0–100% MeCN for 100 min, and 100% MeCN for 20 min) at
a flow rate of 20 mLminꢁ1. UV/Vis wavelength used for the detec-
tion was performed at 330 and 235 nm.
procedure.[24]
Compound 16
TFA (2.78 mL, 35.9, 3 equiv) and N-methylmaleimide (8 mg,
72.0 mmol, 6 equiv) were added successively to a solution of azaph-
thalimide 15 (11 mg, 12.0 mmol) in a H2O/THF mixture (72 mL, 1:1).
The solution was stirred at RT for 18 h and directly purified by
semi-preparative RP-HPLC (System G). The product-containing frac-
tions were lyophilized to afford the product (12 mg, 90% yield) as
System E: RP-HPLC (Varian Kromasil C18 column, 10 mm, 21.2ꢂ
250 mm) with MeCN and 0.1% aq. TFA as eluents (0% MeCN for
10 min, 0–100% MeCN for 100 min, and 100% MeCN for 20 min) at
a flow rate of 20 mLminꢁ1. UV/Vis wavelength used for the detec-
tion was performed at 365 and 310 nm.
1
System F: RP-HPLC (Varian Kromasil C18 column, 10 mm, 21.2ꢂ
250 mm) with MeCN and 0.1% aq. TFA as eluents (0% MeCN for
10 min, 0–100% MeCN for 200 min, and 100% MeCN for 30 min) at
a flow rate of 20 mLminꢁ1. UV/Vis wavelength used for the detec-
tion was performed at 355 and 235 nm.
a yellow oil. H NMR (300 MHz, [D4]MeOD): d=7.95–7.71 (m, 15H),
7.69 (dd, J=8.7, 3.9 Hz, 1H), 7.01–6.93 (m, 1H), 6.93–6.86 (m, 1H),
6.27 (s, 1H), 4.25 (d, J=4.9 Hz, 2H), 3.75 (s, 2H), 3.68–3.56 (m, 2H),
3.25–3.14 (m, 4H), 3.06 (s, 3H), 2.92–3.82 (m, 2H), 2.67 (s, 3H),
2.24–2.13 (m, 4H), 1.77–1.55 (m, 6H), 1.41–1.26 ppm (m, 4H);
13C NMR (75 MHz, [D4]MeOD): d=176.2, 170.8, 168.7, 163.0, 163.0,
162.9, 156.6, 156.6, 152.2, 146.9, 146.5, 136.4 (d, JPꢁC =2.9 Hz, 3C),
134.9 (d, JPꢁC =10.1 Hz, 6C), 131.6 (d, JPꢁC =12.7 Hz, 6C), 127.6,
122.5, 122.3, 119.7 (d, JPꢁC =86.9 Hz, 3C), 114.5, 114.5, 113.9, 102.7,
System G: RP-HPLC (Varian Kromasil C18 column, 10 mm, 21.2ꢂ
250 mm) with MeCN and 0.1% aq. TFA as eluents (0% MeCN for
5 min, 0–20% MeCN for 20 min, 20–60% MeCN for 80 min, and
60–100% MeCN for 35 min) at a flow rate of 20 mLminꢁ1. UV/Vis
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68.5 (d, JPꢁC =16.5 Hz, 1C), 40.1, 38.1, 37.6, 36.9, 33.2, 30.1, 30.0,
29.0, 26.7, 23.9, 23.5 (d, JPꢁC =3.0 Hz, 1C), 20.0 (d, JPꢁC =53.7 Hz, 1C),
19.5 ppm; IR (neat): n˜ =3363, 2942, 2509, 1672, 1611, 1543, 1438,
1385, 1280, 1201, 1178, 1134, 1114 cmꢁ1; HRMS (ESI+): m/z calcd for
C50H52N4O8P: 867.3523 [M]+; found: 867.3499; HPLC (System QC):
tR =27.3 min, purity 97.5%.
DIEA (2.0m solution in NMP, 18 mL, 104 mmol, 5 equiv) was added,
and the resulting reaction mixture was stirred at RT for 2 h. The re-
action completion was checked by RP-HPLC (System QC). Upon re-
action completion, Et2O was added, the resulting precipitate was
centrifuged and dissolved in DMF (2 mL). Hydroxylamine hydro-
chloride (6.8 mg, 104 mmol, 5 equiv) and imidazole (7.2 mg,
104 mmol, 5 equiv) were added, and the reaction mixture was
stirred overnight at RT. The reaction progress was monitored by
RP-HPLC (System QC). After reaction completion, the mixture was
diluted with aq 0.1% TFA and purified by semi-preparative RP-
HPLC (System B). The product-containing fractions were lyophilized
to give the modified peptide 22 (15.96 mg, 69% overall yield for
the two steps) as a red amorphous powder. LRMS (ESI): m/z calcd
for C47H72N18O14: 1113.2050 [M+H]+; found: 1113.13; UV/Vis (PBS):
lmax =500 nm, HPLC (System QC): tR =24.48 min (93.6% purity).
Synthesis of the fluorogenic tetrazine-based probe 19
Compound 19
BOP ((Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexa-
fluorophosphate) (29.2 mg, 0.066 mmol, 1.01 equiv), benzylamine
tetrazine 17[25] (16.1 mg, 0.0718 mmol, 1.1 equiv), and DIEA (46 mL,
0.26 mmol, 4 equiv) were added to a solution of azaphthalimide 6
(20.0 mg, 0.0653 mmol, 1 equiv) in dry DMF (400 mL), and the reac-
tion was stirred at RT for 2 h. Upon reaction completion, the crude
product was purified by flash-column chromatography (silica gel,
EtOAc 100% to Acetone 100%). The residue was diluted with aq
0.1% TFA and purified by semi-preparative RP-HPLC (System E).
The product-containing fractions were lyophilized to give the prod-
uct (6.6 mg, 21%) as an orange amorphous powder. M.p. 1228C;
1H NMR (300 MHz, CDCl3): d=10.58 (s, 1H), 8.55–8.28 (m, 3H), 7.52
(d, J=8.4 Hz, 2H), 4.39 (d, J=5.9 Hz, 3H), 2.97 (s, 3H), 2.87–2.78
(m, 2H), 2.61 (s, 2H), 2.19 (t, J=7.3 Hz, 2H), 1.71–1.51 (m, 4H),
1.42–1.28 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d=172.3, 168.1,
166.6, 165.4, 159.90, 158.1, 145.1, 130.3, 128.1, 128.1, 127.8, 127.8,
121.0, 79.4, 78.9, 78.5, 41.82, 35.3, 32.2, 28.6, 27.3, 25.1, 23.60,
19.6 ppm; IR (neat): n˜ =3279, 2925, 2852, 1697, 1661, 1607, 1515,
1436, 1348, 1251, 1178 cmꢁ1; HRMS (ESI) m/z calcd for C24H25N7O4:
475.1968 [M+H]+; found: 476.2045; HPLC (System QC): tR =
23.15 min (96.3% purity).
Step 2: Peptide 22 (2.78 mg, 2.3 mmol, 1 equiv) and N-hydroxysuc-
cinimide activated maleimide 10 (0.85 mg, 2.7 mmol, 1.2 equiv)
were dissolved in NMP (40 mL). DIEA (2.0m solution in NMP, 3.2 mL,
18.2 mmol, 8 equiv) was added, and the resulting reaction mixture
was stirred at RT for 2 h 30 min. The reaction progress was moni-
tored by RP-HPLC (System QC). Upon reaction completion, the mix-
ture was diluted with aq 0.1% TFA and purified by semi-prepara-
tive RP-HPLC (System A). The product-containing fractions were
lyophilized to give the maleimide-functionalized peptide 23
(1.0 mg, 34%) as a red amorphous powder. LRMS (ESI+) m/z calcd
for C56H81N19O18: 1308.38 [M+H]+; found: 1308.47; UV/Vis (PBS):
labs(max) =500 nm; HPLC (System QC): tR =25.63 min (87.8% purity).
Step 3: 5-Alkoxyoxazole 12 (0.80 mg, 3.1 mmol, 2 equiv),[16] malei-
mide-modified peptide 23 (2.2 mg, 1.55 mmol, 1 equiv) in NMP
(39 mL), and TFA (0.124 mL, 155 mmol, 1 equiv) were combined and
stirred at RT for 3 h. The reaction progress was monitored by RP-
HPLC (System QC). Upon reaction completion, the mixture was di-
luted with aq 0.1% TFA and purified by semi-preparative RP-HPLC
(System C). The product-containing fractions were lyophilized to
give the fluorogenic probe 24 (1.05 mg, 38%) as a red amorphous
powder. HRMS (ESI+) m/z calcd for C66H95N20O21: 1503.6981 [M+H]+
; found: 1503.7013; UV/Vis (PBS): lmax =500 nm. HPLC (System QC):
tR =26.026 min (>99% purity).
Preparation of the uPA-responsive probe 24
4-(methyl(4-((4-nitrophenyl)diazenyl)phenyl)amino)butanoic
acid 21
NOBF4 (187.7 mg, 1.59 mmol, 1.1 equiv) was added to a solution of
para-nitroaniline (200 mg, 1.45 mmol, 1 equiv) in freshly distilled
CH3CN (2 mL) cooled to 08C. The resulting reaction mixture was
stirred at 08C for 15 min. A solution of 4-(methyl-phenyl-amino)-
butyric acid (308 mg, 1.59 mmol, 1.1 equiv) in freshly distilled
CH3CN (1 mL) was added dropwise, and the resulting mixture was
stirred for 30 min at RT. Aq. 0.1m acetate buffer (pH 5.0) was
added slowly until a precipitate appeared. The red solid was recov-
ered by filtration and washed with a mixture of H2O/CH3CN (1:1)
and Et2O. The crude product was purified by flash column chroma-
tography (silica gel, cyclohexane/EtOAc 60:40–40:60–0:100 then
EtOAc/MeOH 90:10). The product (108 mg, 22%) was isolated as
Fluorogenic bioorthognal reaction between norbonene and
tetrazine–azaphthalimide conjugate 19
A solution of norbornene (5 mL, 360 mm in DMSO) was added to
a 10 mm solution of fluorogenic probe 19 in PBS (45 mL, 0.1m
pH 7.4) in the ultra-micro quartz cell. After excitation at the desired
wavelength (420 nm), the fluorescence emission at 520 nm was si-
multaneously monitored over time, with measurements recorded
every 0.1 s. Emission spectra of the probe were recorded before
and after the reaction to determine the quenching efficiency (QE),
which was calculated on the basis of the following equation: QE=
100ꢂ[1ꢁ(fluorescence emission intensity of the probe)/(fluores-
cence emission intensity of the probe after compete reaction of
tetrazine moiety)]
1
a red powder. M.p. 1948C; H NMR (300 MHz, [D6]DMSO): d=8.36
(d, J=9.1 Hz, 2H), 7.93 (d, J=9.1 Hz, 2H), 7.85 (d, J=9.1 Hz, 2H),
6.90 (d, J=9.1 Hz, 2H), 3.55–3.44 (t, J=7.2 Hz, 2H), 3.08 (s, 3H),
2.37–2.17 (t, J=7.2 Hz, 2H), 1.91–1.67 ppm (q, J=7.2 Hz, 2H);
13C NMR (75 MHz, [D6]DMSO): d=174.2, 156.3, 152.6, 146.8, 142.7,
126.0, 125.0, 122.5, 111.6, 50.9, 38.3, 30.7, 21.9 ppm; UV/Vis (DMSO,
258C): lmax abs=500 nm; IR (neat): n˜ =3285, 2921, 2107, 1691,
1601, 1506, 1422, 1289, 1214, 1024, 858, 823, 755, 685, 534,
In vitro enzymatic assays - fluorescence bioassays
410 cmꢁ1
.
A 100 mm solution of fluorogenic peptide 24 was prepared in PBS
(45 mL, 0.1m pH 7.4) and transferred into the ultra-micro quartz
cell. uPA solution (10 mL, 1.2 U, 25 mg in 100 mL of buffer: 500 mm
Tris·HCl+1.0m NaCl+1% PEG 6000+2.0m mannitol) was added,
and the resulting mixture was incubated at 37.58C for 30 min.
After excitation at the desired wavelength (420 nm), the fluores-
cence emission at 520 nm was monitored over time, with measure-
uPA-responsive probe 24
Step 1: The TFA salt of the heptapeptide 9 (20 mg, 18.7 mmol,
1 equiv), the quencher 21 (8 mg, 23.4 mmol, 1.1 equiv), and PyBOP
(12.0 mg, 23.4 mmol, 1.1 equiv) were dissolved in NMP (280 mL).
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Keywords: azaphthalimide
spectroscopy · ligation · oxazole
·
Diels–Alder
·
fluorescence
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Published online on && &&, 0000
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FULL PAPER
&
Fluorescence Probes
K. Renault, L.-A. Jouanno, A. Lizzul-Jurse,
P.-Y. Renard, C. Sabot*
&& – &&
Fluorescent ligation: The fluorogenic
reaction of oxazoles with maleimides
and its subsequent application for the
construction of FRET probes is reported.
Fluorogenic Behaviour of the Hetero-
Diels–Alder Ligation of
5-Alkoxyoxazoles with Maleimides
and their Applications
Chem. Eur. J. 2016, 22, 1 – 11
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