Dramatic increase of quench efficiency in "spacerless" dimaleimide fluorogens

Article abstract of DOI:10.1039/c0ob00455c

In this post-genomic era, new techniques are needed to cope with the task of assigning functional roles to the huge number of identified putative gene products. We have developed a minimalist labelling strategy based on the use of synthetic fluorogenic probe reagents that fluoresce only after their reaction with a target peptide sequence. The probe reagents have fluorescent cores and bear two maleimide groups, such that their latent fluorescence is quenched by a photoinduced electron transfer (PET) to the pendant maleimide groups, until both of these groups undergo a specific thiol addition reaction. The efficiency of the fluorescence quenching is critical to the practicality of this labelling method, and has been predicted to be related to the intramolecular distance between the fluorophore and the maleimide groups. We have conducted the first direct test of this hypothesis by preparing a series of novel fluorogens that differ only by the spacer moiety separating their coumarin fluorophore and their dimaleimide fragment. A striking correlation was observed between intramolecular distance and the fluorescence enhancement (FE) observed after reaction with two equivalents of thiol. Guided by this observation, we then designed 'spacerless' fluorogens, of which a dansyl derivative shows an FE ratio of >300, the largest recorded for dimaleimide fluorogens. The trends observed herein provide valuable lessons for subsequent fluorogen design, and the novel fluorogens developed in the course of this study are currently being applied to protein labelling applications.

Full text of DOI:10.1039/c0ob00455c

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PAPER
Dramatic increase of quench efficiency in “spacerless” dimaleimide
fluorogens†
Karine Caron, Virginie Lachapelle and Jeffrey W. Keillor*
Received 19th July 2010, Accepted 4th October 2010
DOI: 10.1039/c0ob00455c
In this post-genomic era, new techniques are needed to cope with the task of assigning functional roles
to the huge number of identified putative gene products. We have developed a minimalist labelling
strategy based on the use of synthetic fluorogenic probe reagents that fluoresce only after their reaction
with a target peptide sequence. The probe reagents have fluorescent cores and bear two maleimide
groups, such that their latent fluorescence is quenched by a photoinduced electron transfer (PET) to the
pendant maleimide groups, until both of these groups undergo a specific thiol addition reaction. The
efficiency of the fluorescence quenching is critical to the practicality of this labelling method, and has
been predicted to be related to the intramolecular distance between the fluorophore and the maleimide
groups. We have conducted the first direct test of this hypothesis by preparing a series of novel
fluorogens that differ only by the spacer moiety separating their coumarin fluorophore and their
dimaleimide fragment. A striking correlation was observed between intramolecular distance and the
fluorescence enhancement (FE) observed after reaction with two equivalents of thiol. Guided by this
observation, we then designed ‘spacerless’ fluorogens, of which a dansyl derivative shows an FE ratio of
>300, the largest recorded for dimaleimide fluorogens. The trends observed herein provide valuable
lessons for subsequent fluorogen design, and the novel fluorogens developed in the course of this study
are currently being applied to protein labelling applications.
pendant protein may affect the native biology of the labelled
protein of interest.
Introduction
The 20th century has witnessed the emergence of modern ge-
nomics, allowing the sequencing and study of the genomes of
entire organisms, leading to the characterization of a considerable
number of gene products. The fluorescent labelling of proteins has
proven to be a powerful approach to accomplish this task in the
sense that it allows the monitoring of the dynamic processes of
their synthesis and degradation as well as their localization and
interactions with different proteins.1–3
In general two approaches have been taken in the fluorescent
labelling of specific proteins. The first involves the genetic fusion
of the protein of interest with another protein – one that is
either intrinsically fluorescent, or an enzyme that can be labelled
with a fluorescent irreversible inhibitor. For example, fusion with
jellyfish green fluorescent protein (GFP) has seen particularly
broad application,2,4 as has the fusion of enzymes such as phos-
phopantetheine transferase (PPTase),5,6 O6-alkylguanine-DNA
alkyltransferase (AGT)7–9 or mutant haloalkane dehalogenase
(HALO).10 In all these cases, however, the steric bulk of the
The second general approach involves the fusion of a short
peptidic tag to the protein of interest that can either be modified
enzymatically or react specifically with fluorescent labelling agents.
Transglutaminase, 11 biotin ligase,12 lipoic acid ligase,13 and protein
farnesyl transferase14 are examples of enzymes used in this way,
while the FlAsH labelling agents15,16 represent an example of
the small molecule-based labelling of a short peptide tag. This
labelling method has seen widespread application, although some
drawbacks have been noted, including the inherent toxicity of the
organoarsenic compounds employed17 and persistent background
staining.18
In order to address some of the disadvantages of existing
methods, we have developed a complementary protein labelling
technique based on the fluorogenic addition of a dimaleimide
probe to a short a-helical sequence fused to the protein of
interest.19,20 The latent fluorescence of the probe is restored upon
reaction of both maleimide groups21 with the two cysteine residues,
leading to the fluorescent labelling of the protein bearing the target
peptidic sequence (Scheme 1). Thus far, this method has shown
very low cytotoxicity and good selectivity, both representing
important advantages over existing complementary methods.17,18
Although maleimide derivatives have been used for several years
as fluorescent thiol-reactive probes,22–24 little attention has been
given to their mechanism of fluorescence quenching. Our original
De´partement de chimie, Universite´ de Montre´al, CP 6128, Succursale
centre-ville, Montre´al, Que´bec, H3C 3J7, Canada. E-mail: jw.keillor@
umontreal.ca
† Electronic supplementary information (ESI) available: NMR and fluo-
rescence spectra of synthetic intermediates. See DOI: 10.1039/c0ob00455c
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Scheme 1
Chart 1
Scheme 2 Fluorescence quenching by Photoinduced Electron Transfer (PET).
approach to the preparation of such compounds involved the
linear synthesis of a series of aromatic compounds bearing two
maleimide groups attached directly to the fluorescent core.19 The
quench efficiencies measured for these compounds were excellent,
but their low quantum yields and difficult preparation limited
their practical application. Subsequently, a second generation of
dimaleimide fluorogens was prepared by a convergent synthetic
strategy whereby a dimaleimide moiety is linked to a fluorophore
via an intervening spacer.20,21 One of the resulting fluorogens (1,
Chart 1) was used in a photophysical study that demonstrated
that maleimide-mediated quench proceeds via a photoinduced
electron transfer (PET) mechanism (Scheme 2).21 According to
this mechanism, the intramolecular transfer of an electron from
the excited state of the fluorophore to the LUMO of the maleimide
group results in attenuated fluorescence due to non-radiative
relaxation. However, after the maleimide group undergoes its
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Scheme 3 Synthesis of dimaleimide moiety 4.
well-known thiol addition reaction, the LUMO of the resulting
succinimide group is higher in energy, and electron transfer from
the fluorophore excited state is no longer thermodynamically
favoured. As a result, relaxation occurs directly to the ground
state with accompanying fluorescent emission.21
molecule thiols such as glutathione.20 Therefore, the dimaleimide
fragment used in this study was designed to bear two such 3-
methylmaleimide groups. As shown in Scheme 3, dimaleimide
fragment 4 was prepared from 3,5-diaminobenzoic acid and
citraconic anhydride. Since our previously published procedure20
we have optimised conditions for the synthesis of this fragment. In
particular, the maleimide ring closure can be effected reproducibly
and with excellent yield in the presence of ZnCl2 and HMDS
(Scheme 3) rather than with the commonly used acetic anhydride
and sodium acetate.20,26–28
Coumarin was chosen as the fluorophore in the present
series of fluorogens for a number of reason, both synthetic and
photophysical. For this study, the blue fluorescence of coumarin
was deemed to be useful, since the relatively high energy of the
corresponding excited state signifies that electron transfer to the
maleimide LUMO will be thermodynamically favoured, according
to the Rehm–Weller equation.29 Although coumarin derivatives
are well-known and have been used in fluorescent probes, their
syntheses can be laborious and may suffer from low yields.
Moreover, few coumarin derivatives are characterised by high
quantum yields. One exception is 3-carboxy-7-methoxycoumarin
(5), whose electron donating group at position 7 and electron
withdrawing group at position 3 are apparently responsible for its
intense fluorescence (UF = ~0.8 (H2O)).30 Furthermore, efficient
synthetic routes to these particular coumarin derivatives have
been developed, including the method we employed, namely, the
condensation of Meldrum’s acid with 4-methoxysalicylaldehyde,31
shown in Scheme 4.
The practicality of the application of this fluorogenic reaction
to the fluorescent labelling of proteins is directly related to the
efficiency of the fluorescence quench. Fluorogens that are effi-
ciently quenched display lower background fluorescence, allowing
one to envisage a protein labelling technique that does not require
washing to remove excess labelling agent. To this end, we sought to
study and ultimately improve the efficiency of the quench reaction.
The electron transfer from the fluorophore to the maleimide
group that is critical to the quench mechanism21 can occur
through the intervening covalent bonds or through space. Previous
studies have shown that within a certain distance, through-space
electron transfer is more efficient and the predominant manifold.25
Therefore, the role of the spacer moiety in the tripartite design
of our fluorogens becomes crucial, in conferring a conformation
wherein the pendant fluorophore and maleimide groups are in
close proximity to each other. Herein, we report the synthesis
of a series of coumarin-based dimaleimide fluorogens (2a–d,
Chart 1), where the central spacer moiety was systematically
varied, allowing comparison between fluorogen conformation and
contrast ratio.
Results & discussion
Dimaleimide fragment 4 was then linked to 3-carboxy-7-
methoxycoumarin (5), via a series of diamine spacers. In our
previous photophysical study, ethylenediamine was used as a
short but conformationally flexible linker in the tripartite design.
This served as a point of reference for the spacers used in the
present work, whereas other diamine spacers were chosen to allow
control over the distance between the fluorophore and dimaleimide
moieties. The monoprotection of ethylenediamine (as 6a) can be
Design and synthesis of fluorogens
As mentioned above, a convergent synthetic strategy for the
preparation of dansyl fluorogens was conceived in a previous
study and shown to be an efficient way to provide access to series
of fluorogens.21 In more recent work, we have shown that the 3-
methylmaleimide group allows for better kinetic discrimination
between a target protein and adventitious reaction with small
Scheme 4 Synthesis of coumarin fluorophore 5.
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Scheme 5 Synthesis of coumarin fluorogens 2a–d.
accomplished in excellent yield using a ten-fold excess of diamine
over di-tert-butyl carbonate (Boc2O). However, this approach is
impractical for the other, more expensive, diamines used in this
study. Therefore, the latter diamines were monoprotected by slow
addition of one equivalent of di-tert-butyl carbonate to a dilute
solution of diamine, giving compounds 6b–d in fair to good yields.
Fluorophores 7a–d were then prepared in a convergent manner
by coupling fluorophore 5 to spacers 6a–d in the presence of
EDC, HOBt and NEt3, as shown in Scheme 5. After removal
of the Boc group with an excess of TFA, subsequent coupling with
dimaleimide moiety 4 under the same conditions gave fluorogens
2a–d.
Compounds 2a–d incorporate, as spacers, the simplest diamines
that are commercially available for the practical synthesis of fluo-
rogens according to our previously published tripartite design.21 In
order to study labelling agents having even shorter fluorophore–
maleimide distances, we were obliged to significantly alter our
fluorogens. For example, the spacer moiety could be eliminated
altogether if the dimaleimide fragment were re-designed to present
an amine group, to which a fluorophore could be attached directly.
Initially we envisioned designing an aniline-based dimaleimide
fragment and attaching it directly to coumarin 5. However, it
has been shown previously that the quantum yield of coumarin
derivatives bearing a phenyl ring via a conjugated amide group
at position 5 suffer from drastically reduced quantum yields.32
For this reason, a benzylamine-based dimaleimide fragment was
designed instead.
As shown in Scheme 6, 3,5-dinitrobenzyl alcohol 8 was prepared
by reduction of the corresponding carboxylic acid and subse-
quently converted into protected amine 9 by using phthalimide as
a nucleophile under Mitsonobu conditions. Subsequent exchange
of protecting groups gave Boc-protected amine 10. Treatment of
10 with TFA and coupling of the resulting free amine with 7-
methoxycoumarin-3-carboxylic acid resulted in coumarin deriva-
tive 11, which was then submitted to catalytic hydrogenation.
Diamine 12 was then allowed to react with citraconic anhydride
leading to the dimaleimide fluorogenic probe 13.
Quench efficiency
The effects of the spacer moieties in fluorogens 2a–d and
13 were then determined by measurement of the increase in
fluorescence intensity upon reaction with two equivalents of
thiol. The conditions for this fluorogenic reaction were first
optimised. The hydrophilic thiol 2-mercaptopropionic acid (MPA)
was chosen, owing to the practical convenience of its low volatility
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Scheme 6 Synthesis of benzyl coumarin fluorogen 13.
and aqueous solubility. Given the potential application of these
fluorogens as protein labelling agents, their reaction with MPA
(Scheme 7) and the accompanying fluorescence measurements
was studied in a pertinent aqueous solvent mixture (5% DMSO
in 50 mM HEPES buffer (pH 7.4)). The appropriate fluorogen
concentration for these measurements was also determined by
measuring the range over which the fluorescence was linear,
namely, up to 5 mM. At this concentration it was then determined
that the reaction with 50 eq of MPA at room temperature is
complete after 6 h. High resolution mass spectroscopy was used
to confirm that the final addition product was the expected dithiol
adduct.
Measurement of fluorescence intensity before and after reaction
with excess MPA provided contrast ratios, reported as the
fluorescence enhancement (FE) of each fluorogen in Table 1. Since
little change in excitation or emission wavelengths (lexc = 347 nm;
lem = 404 nm) was observed over the course of the fluorogenic
reaction, it is not surprising that little difference was observed
between FE values based on either fluorescence peak height or
peak area.
Scheme 7 Fluorogenic thiol addition reaction between coumarin fluorogens and MPA.
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Table 1 Effect of maleimide–fluorophore distance on quench efficiency for coumarin fluorogens 2a–d and 13
b
FE a
3.17
Through-space distance /A
Through-bond distance/A
˚
˚
Fluorogen
Y
2a
15.6 0.9
19.9
2b
2c
1.84
5.90
16.8 1.5
22.9
14.1 2.4
19.9
19.9
2d
13
4.76
6.40
13.4 1.4
11.8 1.5
15.5
a Fluorescence enhancement, based on the ratio of fluorescence intensity (peak height) measured at lexc = 347 nm and lem = 404 nm, in 50 mM HEPES
buffer (pH 7.4) containing 5% DMSO. b Mean standard deviation.
In general, the FE values determined for the series of coumarin
fluorogens 2a–d are comparable with those reported previously, for
similar dansyl and FITC-based fluorogens. For example, fluorogen
2a, having an ethylenediamine spacer, can be compared to recently
reported dansyl fluorogen 15 and FITC fluorogen 16 (Chart 2).
Fluorogen 2a displays a fluorescence enhancement (FE) value of
3.17 (Table 1) whereas dansyl fluorogen 15 has an FE of 2.0 and
FITC fluorogen 16 has an FE of 5.8.20 However, while all three of
these fluorogens may have the same spacer, they possess different
fluorophores. This means the differences in their FE values may
be as dependent on the different energies of their excited states
and the thermodynamics of electron transfer to the pendant
maleimide group, according to the Rehm-Weller equation,29
as they are on conformational effects25 within the fluorogens.
Therefore, the series of fluorogens studied herein was prepared
in order to be able to study the effect of spacer conformation
alone.
A spacer that links a fluorophore and a pendant maleimide
group can be thought to affect quench efficiency principally
by the distance that it confers between the two moieties.21,29
While the photoinduced electron transfer (PET) responsible for
fluorescence quenching can occur either through bonds or through
space, the latter is considered to be the more effective manifold
for small molecules such as the fluorogens studied herein.29
Molecular modelling was therefore used to predict the maleimide-
to-fluorophore distance of the coumarin–dimaleimide fluorogens
studied herein. In order to evaluate the effective contribution of
each of the two maleimide groups to quenching as realistically as
possible, the through-space distance for each maleimide group
should be determined separately, within a large population of
potential conformers. Therefore, molecular dynamic calculations
were used to simulate how the conformation of each fluorogen
varies over time. Each of the 11000 molecular dynamic snapshots
obtained over the course of a 110 ps run was then minimized
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Chart 2
semi-empirically (AM1), and the distances between the coumarin
fluorophore and both maleimide groups were measured. The time-
averaged fluorophore–maleimide distance was then calculated for
each fluorophore from the theoretical data. As shown in Table 1,
through-space distance data. This observation is consistent with
the theory that through-space electron transfer is the dominant
electron transfer manifold for PET quenching in such small
molecule fluorogens.29
˚
these through-space distances vary significantly, by up to ~5 A
within the series of fluorogens studied, around an average spacer
Spacerless dansyl fluorogen
˚
length of ~14 A.
Based on the results obtained for the series of coumarin fluorogens,
we designed another ‘spacerless’ fluorogen, designed to show even
greater fluorescence enhancement. First, a dimaleimide aniline
moiety was designed, in order to allow its direct attachment to
a fluorophore without an intermediate spacer moiety. Dansyl
was chosen as an appropriate fluorophore, owing to the syn-
thetic convenience of the sulfonamide coupling reaction and
the potential to make comparisons with previously prepared
dansyl fluorogens.20,21 As shown in Scheme 8, the synthesis of
the dimaleimide aniline fragment was initiated with a Schmidt
rearrangement, leading quantitatively to 3,5-dinitroaniline (17).
This was then submitted to catalytic hydrogenation and a mono
Boc protection, giving triamine 18 as a base scaffold for the new
dimaleimide fragment. This mono-protected phenylenetriamine
was then reacted with excess citraconic anhydride, leading to
the dimaleamic acid derivative which was then cyclised to the
dimaleimide core (19) upon treatment with HMDS and ZnCl2. The
dansyl aniline fluorogen (21) was then obtained by deprotection of
the Boc group with excess TFA and coupling of the corresponding
aniline with dansyl chloride in pyridine.
The effect of this variation of through-space distance on the
measured FE values is equally evident. Also shown in Table 1,
the measured FE values were found to vary between <2 and
>6. Furthermore, these values correlate roughly linearly with
the through-space distances calculated between the maleimide
groups and the pendant coumarin fluorophore (Fig. 1). It is
particularly instructive to consider the results obtained with benzyl
derivative 13. Although it lacks the same diamine spacer structure
of compounds 2a–d, it still possesses the same fluorophore
and dimaleimide moieties, and its FE demonstrates the same
distance dependence. This is consistent with the hypothesis that
fluorophore-quencher distance is a critical parameter for quench
efficiency among fluorogens comprising the same of each of these
two moieties. By way of comparison, the average through-bond
distance between maleimide and fluorophore was also determined,
from the minimized structure of each fluorogen. Although a
similar general trend may be inferred from the through-bond data
also shown in Fig. 1, they are clearly not as closely correlated as the
The FE value of fluorogen 21 was then measured as described
above for the coumarin series. We were pleased to observe
an increase in fluorescence intensity of at least two orders of
magnitude upon thiol addition (Fig. 2). The quench efficiency in
fluorogen 21 is so high, that on a practical level, it is difficult to
distinguish its low fluorescence from background noise, leading
to significant error in the calculation of its FE. However, by
a conservative estimate, its fluorescence intensity increases by a
factor of more than 300-fold upon reaction with MPA.
Presumably, the extraordinary quench efficiency inherent in
fluorogen 21 is due to its unique structure. Of course, its ‘spacerless’
design brings the dimaleimide and dansyl moieties into close prox-
imity, but the conformation of the sulfonamide linkage probably
plays an important role, as well. When molecular modelling was
performed on 21 as for the other fluorogens presented above,
the minimised structure, displayed in Fig. 3, suggests that the
Fig. 1 Correlation between fluorescence enhancement and maleimide–
fluorophore distance (see Table 1).
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Scheme 8 Synthesis of spacerless dansyl fluorogen 21.
Fig. 2 Fluorescence increase upon reaction of fluorogen 21 with two
equivalents of MPA.
Fig. 3 Minimised structure of fluorogen 21, showing proximity of dansyl
fluorophore to maleimide group.
sulfonamide group confers a sharp turn, characterised by an acute
C–S–N–C torsion angle of ~75◦. Furthermore, conjugation of the
aromatic NH group presumably limits rotation around the C–N
bond, resulting in the placement of a maleimide group over the
˚
dansyl fluorophore, within <7 A of its core. Although comparison
interactions that facilitate fluorescence quenching may be favoured
in the most highly populated conformation of 21. The discovery of
such a highly efficient fluorogen has opened the door to a variety
of protein labelling applications as well as the design of future
generations of fluorogens.
of this distance–FE correlation with those shown in Fig. 1 is not
justified, since the fluorophores are different, it is obvious that
the FE ratio of 21 is at least an order of magnitude greater than
what we would have roughly predicted from Fig. 1. Specific orbital
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for C17H13N2O6 [M + H]+: 341.0768. Found: 341.0770. mp 254 ◦C
(dec)
Conclusions
The effect of spacer length and conformation on fluorescence
quenching, studied in the series of coumarin maleimides presented
above, confirmed the importance of through-space distance in
the design of efficient fluorogens. This led to the design of di-
maleimide benzyl amine and aniline derivatives that were coupled
directly to coumarin and dansyl fluorophores, respectively. The
resulting dansyl fluorogen 21 is the most efficiently quenched
dimaleimide fluorogen known and the utility of the dimaleimide
aniline moiety is currently being explored, in conjunction with
other fluorophores, for sensitive protein labelling applications.
Furthermore, these fluorogens offer another aspect for potential
exploitation. Considering the two-point attachment to a target
protein conferred by the dimaleimide moiety, a conformationally
constrained fluorogen would allow for the covalent labelling of
a protein with a fluorophore whose orientation is comparatively
fixed, relative to the protein core. The potential application of such
a labelling technique to the investigation of protein orientation and
dynamics, through polarization techniques,33 is currently being
explored.
7-Methoxycoumarin-3-carboxylic acid (5). To a solution of
2-hydroxy-4-methoxybenzaldehyde (3.0 g, 20 mmol) and 2,2-
dimethyl-1,3-dioxane-4,6-dione (2.89 g, 20 mmol) in EtOH
(10 mL) was added piperidinium acetate (58 mg, 0.4 mmol). The
resulting mixture was stirred at 25 ◦C for 20 min, heated to reflux
for 2 h and then cooled at 0 ◦C for 1 h, allowing precipitation of
the title compound 5 (3.71 g, 16.9 mmol, 84%) as white crystals.
1H-NMR (400 MHz, DMSO-d6) d 8.72 (s, 1H), 7.83 (d, J =
8.7 Hz, 1H), 7.04 (d, J = 2.1 Hz, 1H), 7.00 (dd, J = 2.4 and 8.4 Hz,
1H), 3.89 (s, 3H); 13C-NMR (75 MHz, DMSO-d6) d 165.9, 165.5,
158.5, 158.2, 150.4, 115.1, 114.6, 112.9, 101.5, 57.5; HRMS (ESI)
Calculated for C11H8O5Na [M + Na]+: 243.0266. Found: 243.0264.
mp 169 ◦C
tert-Butyl 2-aminoethylcarbamate (6a). To a solution of ethy-
lene diamine (10 mL, 150 mmol) in dry DCM (50 mL) was added
a solution of Boc2O (327 mg, 15 mmol) in DCM (10 mL). The
resulting mixture was stirred at 25 ◦C for 18 h after which the
reaction mixture was washed with a saturated solution of Na2CO3,
dried over MgSO4 and evaporated under reduced pressure to give
the title compound 6a as a colourless oil (2.24 g, 14 mmol, 94%).
1H-NMR (300 MHz, CDCl3) d 5.09 (bs, 1H), 3.12 (dd, J =
5.8 Hz, 2H), 2.74 (t, J = 5.8 Hz, 2H), 1.61 (s, 2H), 1.38 (s, 9H); 13C-
NMR (75 MHz, CDCl3) d 156.3, 79.2, 43.3, 41.8, 28.4; MS (ESI)
Calculated for C11H22N2O2 [M + H]+: 160.12. Found: 161.13.
Experimental section
Generalities
All synthetic starting materials were purchased from Sigma
Aldrich and used without further purification. Solvents were dried
using GlassContour System (Irvine, CA) columns. Reactions re-
quiring anhydrous conditions were carried out under dry nitrogen
atmosphere employing conventional bench-top techniques. 13C-
NMR and 1H-NMR spectra were recorded on AMXR400 or
AMX300 spectrometers and were referenced to the residual proton
or 13C signal of the solvent. Mass spectra were determined by
FAB+ ionization on an AutoSpec Q spectrometer at the Regional
Mass Spectrometry Centre at the Universite´ de Montre´al. Melting
points (uncorrected) were determined on an EZ-Melt (SRS)
melting point apparatus.
trans-tert-Butyl 2-aminocyclohexylcarbamate (6b). To a so-
lution of trans-1,4-diaminocyclohexane (600 mg, 5.25 mmol)
in MeOH (50 mL) were added a solution of Boc2O (1.14 g,
5.25 mmol) in MeOH (10 mL) and NEt3 (2.42 mL, 17.3 mmol).
The resulting mixture was heated at 55 ◦C for 40 min after which
solvents were evaporated under reduced pressure. The resulting
oily residue was washed with NaH2PO4 1 M (pH 4.2) and the
aqueous phase was washed with ether, then basified (pH 9) and
extracted with CHCl3. The organic phase was then dried over
MgSO4 and evaporated under reduced pressure to give the title
compound 6b as a white paste (786 mg, 3.67 mmol, 70%).
1H-NMR (400 MHz, CDCl3) d 4.37 (bs, 1H), 3.39 (bs, 1H),
2.21–2.28 (m, 1H), 1.99–1.89 (m, 2H), 1.69–1.66 (m, 2H), 1.41
(bs, 9H), 1.38–1.33 (m, 5H); 13C-NMR (75 MHz, CDCl3) d 156.1,
80.0, 50.8, 49.8, 36.2, 33.0, 32.9, 29.2; HRMS (ESI) Calculated for
C11H23N2O2 [M + H]+: 215.1754. Found: 215.1754.
Synthesis
3,5-Di-(3-methylmaleimido)benzoic acid (4). To a solution of
3,5-diaminobenzoic acid (1.0 g, 6.58 mmol) in acetone (40 mL)
was added citraconic anhydride (1.8 mL, 19.74 mmol) and the
resulting mixture was stirred at 25 ◦C for 2 h after which solvents
were evaporated under reduced pressure. The crude solid was
triturated with Et2O, filtered under reduced pressure and used
in the next step without further purification. The dimaleamic
acid intermediate (500 mg, 1.33 mmol) was dissolved in DMF
(5 mL) and then toluene (40 mL) was added, followed by ZnCl2
(544 mg, 3.99 mmol) and HMDS (1.13 mL, 5.98 mmol). The
resulting mixture was heated to reflux for 5 h after which the
solvents were removed by rotary evaporation. The title compound
4 was obtained as an off-white solid after precipitation with 0.1 M
HCl (417 mg, 1.22 mmol, 92%).
trans-tert-Butyl 2-aminocyclohexylcarbamate (6c). See exper-
imental procedure detailed for 6b. A yellow paste was isolated
(400 mg, 0.186 mmol, 32%).
1H-NMR (300 MHz, CDCl3) d 4.49 (s, 1H), 3.12 (m, 1H), 1.70
(m, 2H), 1.44 (s, 9 H), 1.22 (m, 6H); 13C-NMR (75 MHz, CDCl3)
d 157.0, 80.1, 58.6, 56.4, 36.0, 33.7, 29.2, 26.0, 25.9; HRMS (ESI)
Calculated for C11H22N2O2 [M + H]+: 215.1754. Found: 215.1762.
cis-tert-Butyl 2-aminocyclohexylcarbamate (6d). See experi-
mental procedure detailed for 6b. A brown oil was isolated
(397 mg, 0.185 mmol, 57%).
1H-NMR (300 MHz, DMSO-d6) d 7.94 (d, J = 1.8 Hz, 2H), 7.62
(t, J = 1.8 Hz, 1H), 6.83 (q, J = 1.8 Hz, 2H), 2.08 (d, J = 1.8 Hz,
6H); 13C-NMR (75 MHz, DMSO-d6) d 170.4, 169.5, 166.2, 146.2,
132.8, 132.2, 128.2, 127.8, 126.0, 11.0; HRMS (ESI) Calculated
1H-NMR (300 MHz, CDCl3) d 4.98 (s, 1H), 3.60 (s, 2H), 3.00
(m, 2H), 1.58 (m, 5H), 1.43 (s, 9H); 13C-NMR (75 MHz, CDCl3)
d 156.5, 79.8, 52.5, 50.7, 32.9, 29.2, 28.8, 23.8, 21.6; HRMS (ESI)
Calculated for C11H23N2O2: 215.1754. Found: 215.1756.
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3-(2-tert-Butoxycarbonylaminoethyl)-aminocarbonyl-7-metho-
xycoumarin (7a). To a suspension of 7-methoxycoumarin-3-
carboxylic acid 5 (585 mg, 2.66 mmol) in acetonitrile (ACN)
(20 mL) were added NEt3 (0.44 mL, 3.19 mmol), EDC (612 mg,
3.19 mmol) and HOBt (431 mg, 3.19 mmol), followed by a solution
of 6a (638 mg, 3.99 mmol) in ACN (5 mL). The resulting mixture
was stirred at 25 ◦C for 16 h after which solvents were evaporated.
The resulting residue was dissolved in CHCl3 and this solution
was washed successively with saturated Na2CO3 and 0.1 M HCl,
dried over MgSO4 and evaporated under reduced pressure to give
the title compound 7a as an off-white solid (660 mg, 1.82 mmol,
69%).
1H-NMR (400 MHz, CDCl3) d 8.94 (bs, 1H), 8.84 (s, 1H), 7.59
(d, J = 8.4 Hz, 1H), 6.95 (dd, J = 2.4 and 8.8 Hz, 1H), 6.86
(d, J = 2.4 Hz, 1H), 4.98, (bs, 1H), 3.92 (s, 3H), 3.58 (q, J =
5.6 Hz, 2H), 3.38 (q, J = 6.6 Hz, 2H), 1.44 (bs, 9H); 13C-NMR (100
MHz, CDCl3) d 164.7, 162.6, 161.5, 156.5, 155.9, 148.2, 130.8,
114.4, 113.9, 112.1, 100.1, 79.2, 55.9, 40.5, 39.7, 28.2; HRMS (ESI)
Calculated for C18H23N2O6 [M + H]+: 363.1551. Found: 363.1548.
1.20 mmol) in CHCl3 (15 mL) was added TFA (3.69 mL, 48 mmol).
The resulting mixture was stirred at 25 ◦C for 4 h after which
solvents were evaporated under reduced pressure. The resulting
oil was triturated with Et2O to give the corresponding amine as a
TFA salt that was used in the next step without further purification.
To a solution of 4 (489 mg, 1.44 mmol) in ACN were added EDC
(277 mg, 1.44 mmol), HOBt (195 mg, 1.44 mmol), NEt3 (0.20 mL,
1.32 mmol) and a solution of the TFA salt in ACN (5 mL) and
NEt3 (0.20 mL, 1.32 mmol). The resulting mixture was stirred
at 25 ◦C for 18 h after which solvents were evaporated under
reduced pressure. The crude solid was dissolved in CHCl3 and the
resulting solution was washed successively with saturated Na2CO3
and 0.1 M HCl, dried over MgSO4 and evaporated under reduced
pressure to give the title compound 2a as a beige solid (521 mg,
0.892 mmol, 74%).
1H-NMR (400 MHz, CDCl3) d 9.19 (t, J = 5.6 Hz, 1H), 8.98 (s,
1H), 7.90 (d, J = 1.6 Hz, 1H), 7.73 (bs, 1H), 7.62–7.60 (m, 2H),
6.94 (dd, J = 2.8 and 8.8 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H), 6.51
(d, J = 2.0 Hz, 2H), 3.92 (s, 3H), 3.77–3.74 (m, 2H), 3.70–3.67
(m, 2H), 2,19 (s, 6H); 13C-NMR (75 MHz, DMSO-d6) d 171.5,
170.6, 166.1, 165.7, 163.2, 161.9, 157.5, 149.2, 147.2, 137.0, 133.9,
133,6, 132.8, 128.9, 125.8, 116.1, 114.9, 113.4, 101.5; HRMS (ESI)
Calculated for C30H25N4O9 [M + H]+: 585.1616. Found: 585.1610.
mp 125 ◦C
trans-1-tert-Butoxycarbonylamino-4-(7-methoxycoumarin-3-
carbonyl-amino)cyclohexane (7b). See experimental procedure
detailed for 7a. An off-white solid was isolated (282 mg,
0.677 mmol, 75%).
1H-NMR (400 MHz, CDCl3) d 8.82 (s, 1H), 7.58 (d, J = 8.8 Hz,
1H), 6.93 (dd, J = 2.4 and 8.8 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H),
4.43 (bs, 1H), 3.91 (bs, 3H), 3.47 (bs, 1H), 2.09–2.07 (m, 4H), 1.62
(s, 1H), 1.45 (bs, 9H), 1.39–1.26 (m, 5H); 13C-NMR (100 MHz,
CDCl3) d 165.6, 162.6, 162.1, 157.4, 156.1, 149.0, 131.7, 115.6,
114.9, 113.2, 101.1, 80.0, 56.8, 49.7, 48.9, 32.9, 32.8, 32.3, 29.2;
HRMS (ESI): Calculated for C22H28N2O6Na [M + Na]+: 439.1840.
Found: 439.1841.
trans-1-tert-Butoxycarbonylamino-4-(7-methoxycoumarin-3-car-
bonylamino)cyclohexane (2b). See experimental procedure
detailed for 2a. A beige solid was isolated (217 mg, 0.340 mmol,
69%).
1H-NMR (400 MHz, CDCl3) d 8.82 (s, 1H), 7.79 (d, J = 1.6 Hz,
1H), 7.64 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 6.94 (dd,
J = 2.4 and 8.4 Hz, 1H), 6.88 (d, J = 2.4 Hz, 1H), 6.51 (d, J =
1.6 Hz, 2H), 6.06 (bs, 1H), 4.02–3.99 (m, 2H), 3.92 (s, 3H), 2.19–
2.17 (m, 8H), 1.48–1.41 (m, 6H); 13C-NMR (75 MHz, CDCl3) d
170.8, 169.7, 165.9, 165.6, 162.6, 162.1, 157.4, 149.0, 146.9, 137.5,
133.4, 131.8, 128.6, 125.8, 125.6, 115.5, 114.9, 113.2, 101.0, 68.8,
56.9, 49.2, 49.0, 32.3, 26.4, 12.1, 12.0; HRMS (ESI) Calculated for
C34H31N4O9 [M + H]+: 639.2086. Found: 639.2079. mp 194 ◦C
trans-1-tert-Butoxycarbonylamino-2-(7-methoxycoumarin-3-
carbonylamino)cyclohexane (7c). See experimental procedure de-
tailed for 7a. An off-white solid was isolated (612 mg, 1.47 mmol,
97%).
1H-NMR (300 MHz, CDCl3) d 8.82 (s, 1H), 7.58 (d, J = 8.7 Hz,
1H), 6.94 (dd, J = 2.4, 8.7 Hz, 1H), 6.87 (d, J = 2.4 Hz, 1H), 5.01
(d, J = 8.1 Hz, 1H), 3.92 (s, 3H), 3.47 (m, 1H), 2.13 (m, 2H), 1.75
(m, 2H), 1.41 (m, 4H), 1.33 (s, 9H); 13C-NMR (75 MHz, CDCl3) d
165.6, 163.3, 162.2, 157.5, 156.6, 149.0, 131.7, 115.5, 114.8, 113.2,
101.2, 79.8, 56.8, 56.1, 54.0, 33.7, 33.2, 29.1, 25.7, 25.5; HRMS
(ESI) Calculated for C22H29N2O6 [M + H]+: 417.2020. Found:
417.2032. mp 141 ◦C.
trans-1-tert-Butoxycarbonylamino-2-(7-methoxycoumarin-3-car-
bonylamino)cyclohexane (2c). See experimental procedure
detailed for 2a. A beige solid was isolated (122 mg, 0.191 mmol,
47%).
1H-NMR (300 MHz, CDCl3) d 8.96 (s, 1H), 8.88 (d, J = 7.80 Hz,
1H), 7.70 (d, J = 6.9 Hz, 2H), 7.58 (d, J = 8.7 Hz, 1H), 7.45 (m, 1H),
6.85 (dd, J = 2.4 and 8.7 Hz, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.45 (m,
J = 1.8 Hz 2H), 3.98 (m, 1H), 3.83 (s, 3H), 2.31 (m, 1H), 2.31 (m,
1H), 2.13 (s, 6H), 2.03 (m, 2H), 1.45 (m, 6H); 13C-NMR (75 MHz,
CDCl3) d 170.8, 169.7, 165.7, 165.4, 164.9, 162.5, 157.6, 150.5,
146.8, 137.2, 133.3, 133.2, 132.0, 128.4, 126.2, 124.4, 114.9, 113.4,
100.9, 58.2, 56.8, 53.0, 33.0, 32.7, 25.8, 25.1, 12.1; HRMS (ESI)
Calculated for C34H31N4O9 [M + H]+: 639.2086. Found: 639.2087.
mp 212 ◦C
cis-1-tert-Butoxycarbonylamino-2-(7-methoxycoumarin-3-car-
bonylamino)cyclohexane (7d). See experimental procedure de-
tailed for 7a. An off-white solid was isolated (256 mg, 0.615 mmol,
45%).
1H-NMR (300 MHz, CDCl3) d 9.06 (d, J = 7.6 Hz, 1H), 8.85 (s,
1H); 7.59 (d, J = 8.8 Hz, 1H), 6.94 (dd, J = 2.4 and 8.4 Hz, 1H),
6.88 (d, J = 2.4 Hz, 1H), 5.07 (d, J = 7.6 Hz, 1H), 4.39 (m, 1H), 3.92
(s, 3H), 1.86 (m, 1H), 1.71 (m, 2H), 1.56 (m, 5H), 1.43 (s, 9 H); 13C-
NMR (75 MHz, CDCl3) d 165.66, 162.83, 162.77, 157.47, 156.30,
149.20, 131.76, 115.64, 114.89, 113.24, 101.09, 80.16, 77.51, 77.45,
56.85, 50.02, 31.77, 30.17, 29.72, 29.22; HRMS (ESI) Calculated
for C22H29N2O6 [M + H]+: 417.2020. Found: 417.2020. mp 213 ◦C
cis-1-tert-Butoxycarbonylamino-2-(7-methoxycoumarin-3-car-
bonylamino)cyclohexane (2d). See experimental procedure de-
tailed for 2a. A beige solid was isolated (287 mg, 0.450 mmol,
82%).
1H-NMR (300 MHz, CDCl3) d 9.35 (d, 1H, J = 7.8 Hz), 9.04 (s,
1H), 8.80 (s, 1H), 7.98 (m, 1H), 7.90 (d, J = 1.8 Hz, 1H), 7.63–7.60
3-(2-(3,5-Bi-(3-methylmaleimido)-benzoylamino)ethylaminocar-
bonyl)-7-methoxycoumarin (2a). To a solution of 7a (435 mg,
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(m, 3H), 6.99 (dd, J = 1.8 and 8.7 Hz, 1H), 6.90 (d, J = 2.1 Hz,
1H), 6.47 (bs, 2H), 4.53 (bs, 1H), 3.92 (bs, 3H), 3.11 (bs, 1H),
2.19 (bs, 6H), 2.06–1.75 (m, 8H); 13C-NMR (75 MHz, CDCl3) d
170.8, 169.7, 166.1, 165.9, 164.5, 162.9, 157.6, 150.2, 146.8, 137.5,
133.2, 131.8, 128.4, 125.6, 123.9, 115.1, 115.1, 113.3, 101.1, 56.9,
54.7, 50.1, 30.7, 27.8, 24.8, 21.5, 12.0; HRMS (ESI) Calculated for
C34H31N4O9 [M + H]+: 639.2086. Found: 639.2084. mp 165 ◦C
13C-NMR (75 MHz, CDCl3) d 155.8, 148.4, 144.2, 127.1, 117.5,
80.6, 43.4, 28.1; HRMS (ESI) Calculated for C12H15N3O6 [M]-:
297.0963. Found: 297.0966. mp 94 ◦C.
7-Methoxy-3-(3,5-dinitrobenzyl)aminocarbonylcoumarin (11).
TFA (2.77 mL, 36 mmol) was added to a solution of the
Boc-protected 3,5-dinitrobenzyl amine 10 ((267 mg, 0.898 mmol)
in DCM (10 mL). The resulting mixture was stirred at 25 ◦C
for 2 h after which solvents were evaporated. A white solid was
precipitated from the resulting oil upon addition of Et2O, and
used in the next step without further purification. A solution
of the TFA salt in ACN (10 mL) was added to a solution of
coumarin (218 mg, 0.988 mmol), EDC (189 mg, 0.988 mmol),
HOBt (134 mg, 0.988 mmol) and NEt3 (0.26 mL, 1.89 mmol) in
ACN (20 mL). The resulting mixture was stirred at 25 ◦C for 18 h
after which solvents were evaporated under reduced pressure. The
crude was dissolved in CHCl3 and this organic phase was washed
successively with saturated Na2CO3 and 0.1 M HCl, dried over
MgSO4 and evaporated under reduced pressure to give 11 as a
beige solid (298 mg, 0.747 mmol, 83%).
1H-NMR (400 MHz, DMSO-d6) d 9.41 (t, J = 5.6 Hz, 1H), 8.45
(s, 1H), 8.72 (t, J = 2.0 Hz, 1H), 8.65 (d, J = 2.0 Hz, 2H) 7.90 (d,
J = 8.8 Hz, 1H), 7.13 (d, J = 2.4 Hz, 1H), 7.05 (dd, J1 = 2.4, J2 =
8.8 Hz, 1H), 4.75 (d, J = 6.0 Hz, 1H), 3.92 (s, 3H); 13C-NMR (75
MHz, DMSO-d6) d 165.0, 162.7, 161.1, 156.8, 148.8, 148.4, 144.6,
132.1, 128.7, 117.7, 115.0, 114.1, 112.6, 100.8, 56.8, 31.2; HRMS
(ESI) Calculated for C18H13N3O8Na [M + Na]+: 422.0589. Found:
422.0595. mp 233 ◦C.
3,5-Dinitrobenzyl alcohol (8). To a suspension of NaBH4
(2.85 g, 75.4 mmol) in dry THF (40 mL) at 0 ◦C was added a
solution of 3,5-dinitrobenzoic acid (8.0 g, 37.7 mmol) in THF
(20 mL), followed by BF3·OEt2 (12.5 mL, 98.0 mmol). The
◦
resulting mixture was warmed to 25 C and stirred for 1 h after
which the reaction was quenched with 1 M HCl. The aqueous
phase was extracted with DCM and the organic phase was then
washed with saturated Na2CO3. The organic phase was dried over
MgSO4 and evaporated under reduced pressure to give the title
compound 8 as an orange solid (5.01 g, 25.3 mmol, 67%).
1H-NMR (400 MHz, ((CD3)2CO) d 8.91 (t, J = 0.4 Hz, 1H), 8.57
(dd, J = 1.2 and 2.0 Hz, 2H), 4.93 (d, J = 5.2 Hz, 2H), 3.57 (bs,
1H); 13C-NMR (75 MHz, (CD3)2CO) d 149.4, 148.4 (2C), 127.0
(2C), 117.6, 62.8; HRMS (ESI) Calculated for C7H5N2O5 [M -
H]-: 197.0204. Found: 197.0200. mp 89 ◦C.
N-(3,5-Dinitrophenyl)methylphthalimide (9). To a solution of
3,5-dinitrobenzyl alcohol 8 (1.38 g, 6.94 mmol), triphenylphos-
phine (2.19 g, 8.33 mmol) and phthalimide (1.23 g, 8.33 mmol)
in dry THF (40 mL) under N2 atmosphere was added DIAD
(1.64 mL, 8.33 mmol). The resulting mixture was stirred at 25 ◦C
for 48 h after which solvents were evaporated under reduced
pressure. The crude was triturated with EtOAc and filtered under
reduced pressure to give the title compound 9 as a beige solid
(1.33 g, 4.05 mmol, 58%).
1H-NMR (400 MHz, CDCl3) d 8.98 (t, J = 2.0 Hz, 1H), 8.61
(d, J = 2.0 Hz, 2H), 7.93–7.89 (m, 2H), 7.81–7.77 (m, 2H), 5.05
(s, 2H); 13C-NMR (75 MHz, CDCl3) d 168.5, 168.4, 149.6, 141.3,
135.5, 132.5, 129.6, 124.7, 119.3, 41.2; HRMS (ESI) Calculated
for C15H10N3O6 [M + H]+: 328.0564. Found: 328.0568. mp 171 ◦C
7-Methoxy-3-(3,5-di-(3-methylmaleimido)benzyl)aminocarbony-
lcoumarin (12). To a suspension of 11 (80 mg, 0.203 mmol)
in DMF (8 mL) was added Pd/C 10% (50% H2O w/w). The
resulting mixture was stirred under 1 atm H2 at 25 ◦C for 18 h after
which it was filtered over celite. Citraconic anhydride (0.06 mL,
0.609 mmol) was then added to the filtrate and the resulting
mixture was stirred at 25 ◦C for 18 h after which toluene (20 mL)
was added, followed by ZnCl2 (83 mg, 0.609 mmol) and HMDS
(0.13 mL, 0.609 mmol). The resulting mixture was heated to reflux
for 4 h and solvents were evaporated under reduced pressure. The
crude residue was dissolved in EtOAc and the organic phase was
washed successively with 0.1 M HCl and saturated Na2CO3, dried
over MgSO4 and evaporated under reduced pressure to give the
title compound 12 as a beige solid (38 mg, 0.072 mmol, 35%).
1H-NMR (400 MHz, DMSO-d6) d 9.20 (bs, 1NH), 8.86 (s, 1H),
7.95–7.91 (m, 2H), 7.35 (s, 1H), 7.22 (s, 1H), 7.13 (s, 1H), 7.05 (d,
J = 8.0 Hz, 1H), 6.81 (s, 2H), 4.61 (d, J = 5.2 Hz, 2H), 3.91 (s, 3H),
2.07 (s, 6H); 13C-NMR (75 MHz, DMSO-d6) d 171.6, 170.7, 165.8,
163.0, 162.0, 157.5, 149.4, 147.1, 142.3, 133.6, 132.9, 128.8, 126.0,
124.6, 1156.0, 115.0, 113.4, 101.6, 57.5, 43.5, 12.1; HRMS (ESI)
Calculated for C28H22N3O8 [M + H]+: 528.1421. Found: 528.1401.
tert-Butyl 3,5-dinitrophenylcarbamate (10). To a solution of
9 (960 mg, 2.93 mmol) in THF (20 mL) were added hydrazine
(1.14 mL, 23.4 mmol) and p-nitrotoluenesulfonic acid (280 mg,
1.47 mmol). The resulting mixture was heated to reflux for
2 h after which it was cooled down to 25 ◦C and saturated
Na2CO3 was added. The aqueous phase was extracted with CHCl3
and the organic fractions were combined, dried over MgSO4
and evaporated under reduced pressure to give the free 3,5-
dinitrobenzyl amine as a yellow solid that was used without further
purification. A solution of 3,5-dinitrobenzyl amine in MeOH
(20 mL) was treated with Boc2O (1.28 g, 5.86 mmol) and NEt3
(0.62 mL, 4.40 mmol). The resulting mixture was heated to reflux
for 45 min and then stirred at 25 ◦C for 18 h, after which the
solvents were evaporated. The resulting residue was dissolved
in CHCl3 and this organic phase was washed with 0.1 M HCl,
dried over MgSO4 and evaporated under reduced pressure. The
resulting solid was purified by flash chromatography on silica gel
(DCM/20% Hex) to give the title compound 10 as a white solid
(945 mg, 2.34 mmol, 80%).
3,5-Dinitroaniline (17). To a solution of 3,5-dinitrobenzoic
acid (4.0 g, 18.9 mmol) in a mixture of concentrated sulfuric acid
(6 mL), 30% fuming sulfuric acid (18 mL) and CHCl3 (25 mL)
was added sodium azide (1.4 g, 21.7 mmol), in small portions. The
resulting mixture was heated to reflux (80 ◦C) for 3 h and then
cooled to 25 ◦C after which it was poured on ice. An orange solid
was isolated after vacuum filtration (3.25 g, 17.7 mmol, 94%).
1H NMR (300 MHz, CDCl3) d 8.14 (t, J = 1.8 Hz, 1H), 7.64 (d,
J = 1.8 Hz, 2H), 4.39 (s (br), 2H); 13C NMR (75 MHz, CDCl3) d
1H-NMR (400 MHz, CDCl3) d 8.93 (bs, 1H), 8.49 (d, J = 2.4 Hz,
2H), 5.28 (bs, 1NH), 4.52 (d, J = 6.0 Hz, 2H), 1.47 (bs, 9H);
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151.9, 150.1, 113.1, 105.2; HRMS (ESI) Calculated for C6H4N3O4
[M - H]-: 182.0195. Found: 182.0207. mp: 113.1 ◦C.
was purified by flash chromatography on silica gel (Tol/EtOAc
10%) giving 21 as a light green solid (40 mg, 0.074 mmol, 16%).
1H NMR (400 MHz, (CD3)2CO) d 9.71 (s, 1H), 8.56 (d, J =
8.4 Hz, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.39 (dd, J1 = 1.2, 7.2 Hz, 1H),
7.62–7.58 (td, J = 1.2, 7.2 Hz, 2H), 7.28–7.27 (m, 3H), 7.11 (t, J =
2.0 Hz, 1H), 6.63–6.61 (m, 2H); 13C NMR (75 MHz, (CD3)2CO) d
171.3, 170.4, 152.8, 135.4, 134.0, 131.7, 131.5, 130.3, 130.2, 129.6,
128.7, 124.9, 119.8, 119.6, 116.6, 115.9, 46.3, 12.1; HRMS (ESI)
Calculated for C28H25N4O6S [M + H]+ 545.1516. Found: 545.1489.
mp 230 ◦C
tert-Butyl N-3,5-diaminophenylcarbamate (18). A solution of
17 (2.3 g, 12.56 mmol) in THF–MeOH (30/5 mL) in presence of
Pd/C 10% (50% m/m H2O) was stirred at room temperature under
10 atm hydrogen for 24 h after which the solvents were filtered on
celite. The resulting phenylenetriamine was used in the next step
without further purification. The filtrate volume was increased to
200 mL after which a solution of Boc2O (2.7 g, 12.56 mmol) in
THF (20 mL) was added over 30 min. The resulting solution was
heated to reflux for 16 h after which volatiles were evaporated
under reduced pressure. The crude was then purified by flash
chromatography on silica gel (elution gradient DCM–Hex (8 : 2)
to DCM–MeOH (49 : 1)) to give 18 (958 mg, 4.29 mmol, 35%) as
an off-white solid.
1H NMR (400 MHz, CDCl3) d 6.18 (d, J = 2 Hz, 2H), 5.95 (bs,
1H), 5.73 (t, J = 2.0 Hz, 1H), 3.57 (bs, 4H), 1.50 (bs, 9H); 13C NMR
(100 MHz, CDCl3) d 152.5, 148.0, 140.1, 96.8, 96.0, 80.1, 28.2;
HRMS (ESI) Calculated for C11H17N3O2Na [M + Na]+: 246.1218.
Found: 246.1214.
Determination of fluorescence enhancement ratios
Absorbance spectra were recorded at 25 ◦C, with a Cary-100
spectrometer. Emission spectra and fluorescence intensity mea-
surements were recorded at 25 ◦C with a Cary Eclipse fluorometer.
Excitation and emission slits were fixed at 5 nm.
To a 1 to 4 mM solution of fluorogen in DMSO was added 3-
mercaptopropionic acid (50 eq). The resulting mixture was stirred
at 25 ◦C, in the dark for 18 h after which the fluorescence intensities
of a dilution (~4 mM final) in HEPES buffer (pH 7.4) were
recorded. The final fluorescence intensity was then divided by the
initial florescence intensity at the same fluorogen concentration to
give the fluorescence enhancement ratio.
tert-Butyl N-3,5-di-(2-methylmaleimido)phenylcarbamate (19).
Citraconic anhydride (1.56 mL, 17.47 mmol) was added to a
solution of 18 (1.3 g, 5.82 mmol) in CHCl3 (20 mL) and the
resulting mixture was stirred at 25 ◦C for 3 h after which volatiles
were evaporated under reduced pressure. The crude was triturated
with Et2O and filtered under reduced pressure leading to the
dimaleamic acid as a beige solid (2.59 g, 5.80 mmol, quantitative)
that was used in the next step without further purification. The
dimaleamic acid (400 mg, 0.894 mmol) and ZnCl2 (365 mg,
2.68 mmol) were dissolved in toluene–DMF (40 : 5 mL) before a
dilute solution of HMDS (0.84 mL, 4.02 mmol) in toluene (5 mL)
was added over 20 min. The resulting mixture was then heated
to reflux for 4 h after which the volatiles were evaporated under
reduced pressure. The resulting residue was dissolved in EtOAc
and washed successively with 0.1 M HCl and saturated Na2CO3.
The crude product was then purified by flash chromatography on
silica gel (Tol/EtOAc 5%) giving 19 as an off-white solid (780 mg,
1.90 mmol, 53%).
Molecular modelling
The geometry of each fluorogen was initially optimized in vacuo,
using the AM1 semi-empirical method and the Polak–Ribiere
conjugate gradient algorithm, with convergence being determined
-1
by an RMS gradient of <0.1 kcal A mol-1, according to the
˚
standard parameters of the software HyperChem Professional 7.5.
Molecular dynamic simulations were then performed in vacuo by
heating the system from 300 K to 1000 K over a 10 ps period,
followed by a 100 ps constant temperature simulation. These
conditions allowed the system to explore such conformational
equilibria as cis/trans amide bonds and boat/chair cyclohexane
rings. At each 10 fs interval, the distance was recorded between
the central carbon atom C9 of the coumarin ring and the tertiary
carbon atom C3 of each of the two maleimide groups. According to
the orbital density calculated by the AM1 semi-empirical method,
these atoms feature prominently in the fluorophore excited state
and the maleimide LUMO, thereby providing a reasonable
estimate of the distance of electron transfer according to the
PET quench mechanism. Final conformations were optimized
by subsequent minimization according to the method described
above. For the calculation of the through-bond distance of each
fluorogen, the lengths of the bonds between all atoms from
coumarin C9 to maleimide C3 were added.
1H NMR (400 MHz, CDCl3) d 7.42 (d, J = 2.4 Hz, 2H), 7.12
(t, J = 2.4 Hz), 6.59 (bs, 1H), 6.43 (q, J = 2.4 Hz, 2H), 2.12 (q,
J = 2.4 Hz, 6H), 1.47 (bs, 9H); 13C NMR (75 MHz, CDCl3) d
170.0, 168.9, 152.2, 145.7, 139.5, 132.5, 127.4, 116.5, 114.1, 80.9,
28.1, 11.0; HRMS (ESI) Calculated for C21H21N3O6Na [M + Na]+:
434.1331. Found: 434.1323. mp 179 ◦C
3,5-Di(3-methylmaleimido)anilinium trifluoroacetate (20).
A
solution of 19 (780 mg, 1.90 mmol) in CHCl3 (10 mL) was treated
with TFA (5.9 mL, 76 mmol) at 25 ◦C for 3 h after which the
volatiles were evaporated. An off-white solid was then precipitated
with Et2O (805 mg, 1.89 mmol, Q). The TFA salt 20 was used in
the next step without further purification.
Acknowledgements
N-Dansyl-3,5-di-(3-methylmaleimido)aniline
(21). Dansyl
chloride (139 mg, 0.517 mmol) in pyridine (3 mL) was added to
a pyridine solution of 20 (200 mg, 0.517 mmol). The resulting
mixture was stirred at 25 ◦C for 18 h after which the volatiles were
evaporated under reduced pressure. The resulting oil was taken
back in CHCl3 and the organic phase was washed with aqueous
saturated Na2CO3, dried over MgSO4 and evaporated. The crude
This work was supported by the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC) and the Canadian
Institutes of Health Research (CIHR). KC acknowledges a
postgraduate bursary from the Fonds Que´becois de Recherche
sur la Nature et les Technologies (FQRNT) and VL acknowledges
NSERC for a summer internship bursary.
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