Fluorescent properties and resonance energy transfer of 3,4-bis(2,4-difluorophenyl)-maleimide

Article abstract of DOI:10.1039/c2ob25981h

A fluorescent compound 3,4-bis(2,4-difluorophenyl)-maleimide 1 from the 3,4-diaryl-substituted maleimides was synthesized and determined to have a Stokes shift of 140 nm (λ_abs 341 nm, λ_em 481 nm), a high fluorescent quantum yield (Φ_fl 0.61) and an extinction coefficient ε₍₃₄₀₎ of 48 400 M^-1 cm ^-1 in dichloromethane. For the first time we demonstrated the successful implementation of a 3,4-diaryl-substituted maleimide molecule as a donor component in FRET experiments. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25981h

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PAPER
Fluorescent properties and resonance energy transfer of
3,4-bis(2,4-difluorophenyl)-maleimide†
Katya P. Nacheva,a William A. Maza,a David Z. Myers,a Frank R. Fronczek,b Randy W. Larsena and
Roman Manetsch*a
Received 21st May 2012, Accepted 2nd August 2012
DOI: 10.1039/c2ob25981h
A fluorescent compound 3,4-bis(2,4-difluorophenyl)-maleimide 1 from the 3,4-diaryl-substituted
maleimides was synthesized and determined to have a Stokes shift of 140 nm (λabs 341 nm, λem 481 nm),
a high fluorescent quantum yield (Φfl 0.61) and an extinction coefficient ε(340) of 48 400 M−1 cm−1 in
dichloromethane. For the first time we demonstrated the successful implementation of a 3,4-diaryl-
substituted maleimide molecule as a donor component in FRET experiments.
maleimides and the development of an efficient organic light-
Introduction
emitting diode (OLED) based upon a naphthylphenylamino sub-
stituted N-methyl-3,4-diphenyl-maleimide.4 The ongoing effort
Fluorescent molecules find important applications as sensor
elements, biomolecular probes, and substrates in material and
life sciences. Despite the large variety of available fluorescent
dyes, new fluorophoric systems possessing a large Stokes shift, a
high quantum yield, and a high photostability are of continued
interest.1 Well known fluorophores such as cyanines, tetramethyl
rhodamines (TMR), and fluoresceins are characterized by broad
excitation and emission ranges and small Stokes shifts (less than
30 nm in some cases), which limit their application in Förster
resonance energy transfer (FRET) systems due to partial absorp-
tion of the incident light by the FRET acceptor partner.2a At the
same time while Alexa 350 and Alexa 430 dye are characterized
with a high Stokes shift, greater than 70 nm, they exhibit lower
extinction coefficient.2b In addition, fluorescein conjugates
appended to biological molecules are quite often photolabile and
their fluorescence is significantly quenched prior to photodisso-
ciation. In contrast, the use of the diaryl-substituted maleimides
(DMs) as fluorescence probes in bioscience applications have
not been extensively explored. Despite their promising photo-
physical characteristics such as high fluorescent quantum yields
(Φfl > 0.10), good extinction coefficients (ελ > 5000 M−1 cm−1),
and large Stokes shifts (Δ > 90 nm), the DM molecules have
been investigated primarily for material science applications.3
Recent reports demonstrated the design and the application of
fluorescent metal sensors based on monoindole-substituted
to generate small-molecule fluorescent probes with high fluore-
scent quantum yield, a large Stokes shift and high photostability,
prompted us to investigate the photophysical characteristics of a
new fluorescent dye from the DM family, 3,4-bis(2,4-difluoro-
phenyl)-maleimide 1, and its functionalized derivatives. The
simple and efficient synthetic route to yield 1 and the convenient
way to functionalize that moiety with appropriately installed
chemical handles, makes this fluorophore easily accessible.
Herein we present the identification and spectroscopic character-
ization of 3,4-bis(2,4-difluorophenyl)-maleimide 1 and its first
reported use as a donor component in FRET systems.
Results and discussion
Photophysical properties
The analysis of the photophysical properties of 1 revealed a high
extinction coefficient ε(340) 48 400 M−1 cm−1, a large Stokes
shift (Δ) of 140 nm in MeOH (λabs 341 nm; λem 481 nm), and a
very good fluorescence quantum yield in DCM (Φfl 0.61)
(Fig. 1). It is important to note that when compared to the
reported DM molecules (ε(λ) 2180 to 14 000 M−1 cm−1 and Φfl
0.02 to 0.52), 1 exhibits a higher extinction coefficient (ε(λ)
13 490 to 48 400 M−1 cm−1) and excellent quantum yield in a
variety of solvents (Φfl 0.08 to 0.61) in addition to the unusually
large Stokes shift.
These advantageous spectral characteristics render 1 as a suit-
able donor component in a FRET system, exemplified in the
large Stokes shift which is essential for an efficient energy trans-
fer between the donor and acceptor with a minimal direct exci-
tation of the donor. Therefore, having a donor with an excitation
maxima being sufficiently apart from the excitation acceptor,
aDepartment of Chemistry, University of South Florida, 4202 East
Fowler Ave, CHE 205, Tampa, FL 33620, USA.
E-mail: manetsch@.usf.edu
bDepartment of Chemistry, Louisiana State University, Baton Rouge,
LA 70803, USA
†Electronic supplementary information (ESI) available. CCDC
XXXXXX. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c2ob25981h
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results in a more accurate measurement of the energy transfer
phenomena.
obtained by substituting the DM fluorophore with various func-
tionalities tethered to the nitrogen. Much like the parent com-
pound 1, the analogs 1a, 1b, and 1c display high fluorescence
quantum yields, high molar extinctions (ε(λ) 5990 to 23 000),
and a large Stokes shifts (Δ 115 to 143 nm depending on
solvent).
Bathochromic shifts were observed in the emission spectra for
1 and its derivatives when moving from nonpolar to polar sol-
vents (Table 1). The largest bathochromic shift in the emission
spectra, as well as the largest values for the Stokes shift for all
compounds was recorded in the presence of the protic polar
solvent methanol (Δ 135 nm for 1c to Δ 143 nm for 1b) while
the lowest values were observed for the more polar aprotic
solvent CH3CN (Δ 122 nm for 1b to Δ 130 nm for 1). In con-
trast, the absorption maxima of 1 and 1a–1c display only small
differences between solvents. This observation suggests that
hydrogen bonding between 1 and the methanol solvent stabilizes
the singlet excited state.
An efficient one step synthesis of 1 was achieved by allowing
(2,4-difluoro-phenyl)-acetonitrile to react with iodine and
sodium methoxide at −85 °C (Scheme 1).3 The exact structure
of 1 was confirmed by 1H-, 13C-, 19F-NMR spectroscopy, and by
high resolution mass spectrometry. An X-ray diffraction structure
was also obtained, demonstrating that 1 has noncrystallographic
C2-symmetry with the two difluorophenyl rings not being co-
planar to the maleimide ring plane (Fig. 2).
In an attempt to implement the fluorescent probe into a FRET
system, we generated derivatives 1a, 1b, and 1c containing
chemical handles, enabling the linkage to a FRET partner of
interest.1 Conceivably, such types of derivatives were easily
Seliskar and McGlynn5a characterized the lowest lying singlet
state of the unsubstituted maleimide as a 11A1 → 11B2 transition
of primarily n → π* character. However, it is well known that
transitions to n → π* states are strongly dependent upon the
nature of the solvent while transitions to π → π* states are not as
strongly influenced.5b Thus, the fact that the absorption maxima
of 1, 1a–1c do not exhibit a significant solvent dependence
would suggest that the excitation is largely of π → π* origin
while the lowest energy emitting state is of n → π* character
thus accounting for the significant solvent dependence of the
Stokes shift. The re-ordering of the excitation state may be a
Fig. 1 Normalized absorption and emission of 1 in DCM.
Scheme 1 Synthesis of 3,4-bis(2,4-difluorophenyl)-maleimide 1 and
the analogs 1a, 1b, and 1c.
Fig. 2 X-ray crystal structure of 1 with 50% ellipsoids.
Table 1 Solvent dependent absorption and emission maxima of compounds 1, 1a–1c
1
1a
1b
1c
b
ε
λabs/λfl a (Stokes)
λabs/λfl a (Stokes)
λabs/λfl a (Stokes)
λabs/λfl a (Stokes)
Φfl
Hexanes
Toluene
DCM
CH3OH
CH3CN
1.89
2.38
8.93
32.7
37.5
345/460 (115)
345/463 (118)
345/468 (123)
341/481 (140)
338/468 (130)
334/445 (111)
331/448 (117)
332/454 (122)
332/469 (137)
325/452 (127)
346/465 (119)
346/469 (123)
348/467 (119)
340/483 (143)
342/464 (122)
342/461 (119)
346/464 (118)
350/469 (119)
346/481 (135)
344/469 (125)
0.083
0.06
0.61
0.313
0.296
a Absorption and emission spectra were recorded in 5 × 10−6 M solutions, λex 340 nm. b Quantum yield of 1 was measured against perylene.
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Fig. 3 Normalized emission spectra of 1 (DCM, 1 × 10−5 M) after
irradiation with Xe source for 6–18 hours, (λexc = 340 nm).
result of the phenyl substitution on the parent maleimide ring
and this is currently under investigation.
pH sensitivity and photobleaching stability
The pH sensitivity and stability towards photobleaching of 1
were explored as such preconditions are important when select-
ing a fluorophore for general biological or FRET applications.
First, the photobleaching test was performed by monitoring the
intensity of the absorption and fluorescence spectra of 1 in DCM
irradiated by a broad spectrum Xe source (150 W) for an
extended period of time (6–18 hours). This experiment indicated
that 1 has a very good photo-stability with a negligible loss of
fluorescence (∼2%) after 18 hours of illumination (Fig. 3).
Maleimides are known to undergo intersystem crossing
leading to formation of an excited triplet state.5,6 If the energy of
the triplet of 1 is estimated to be close to that of the parent malei-
mide structure, the quantum yield of triplet formation of 1 in
CH3CN is ∼0.37 and increases with less polar solvents.5 The
low photobleaching susceptibility of 1, despite the substantial
efficiency of triplet formation, may be an intrinsic property
related to the polyfluorination of the aromatic rings. It has been
reported that substitution by heavy atoms, such as fluorine in the
case of 1 and its derivatives, significantly improves the photo-
bleaching stability of cyanine dyes substituted with sulfonyl
groups despite the sulfonyl group’s propensity to increase triplet
yields.7
Fig. 4 Fluorescence pH dependence of 1 under (A) acidic conditions,
TFA (1 in CH3CN, 5 × 10−6 M) and (B) basic conditions, 2,6-lutidine
(1 in CH3CN, 5 × 10−6 M), λexc = 340 nm.
The quenching effect was even more pronounced when a
stronger base such as triethylamine was titrated to 1. The
decrease in the fluorescence intensity of 1 at higher pH is attribu-
ted to the deprotonation of the bisamido group and the gener-
ation of a non-fluorescent monoanion. In addition, the fact that
the alkylated analog 1b displayed higher stability to basic media
confirmed the above mechanism of a base-induced reduction in
fluorescence.† The fluorescence reduction was found to be
reversible upon treatment of the anion with acid.† This suggests
that the n → π* transition of the excited singlet state to the
ground state primarily involves the lone pair on the nitrogen
while also confirming the role of hydrogen bonding in stabilizing
the excited state.
Due to the low aqueous solubility of 1, the pH dependence of
the fluorescence could not be accurately recorded in aqueous
media. Therefore, experiments to determine a potential pH
dependence were carried out in organic solvents by analyzing
the photophysical properties of 1 under acidic (TFA) and basic
(2,6-lutidine) environments. It was observed that the spectral
profile of 1 was not influenced by the acidic media (Fig. 4A),
while a steady decrease of the fluorescence intensity was
observed when 1 was titrated with 2,6-lutidine (Fig. 4B). Rehm–
FRET system with quencher moiety as an acceptor
Considering the advantageous photophysical properties of DMs
1 and 1a–1c, we evaluated 1c as a donor component in a FRET
peptide substrate of β-secretase, a key enzyme responsible for
Alzheimer’s disease.9 The sequence of the peptide was derived
from the β-secretase cleavage site of the Swedish APP mutation
(previously reported by Anna Spec). The envisioned FRET
peptide contains DM donor moiety and an acceptor (quencher)
motif allowing for depopulation of the donor excited state via an
intramolecular FRET mechanism. The peptide synthesis was per-
formed by using standard Fmoc chemistry on solid phase with
subsequent coupling of the donor–acceptor pair.† The fluoro-
phore donor 1c was appended to the N-terminus, and the
Weller analysis using Eox(lutidine) of 1.89 V (SCE) and Ered
-
(maleimide) = −1.03 V (SCE) and 2.65 V for the energy of the
singlet excited state of 1 in DCM (λmax = 468 nm) results in a
positive potential ∼0.3 V indicating the mechanism of quenching
is not electron transfer between 1 and 2,6 lutidine.8
7842 | Org. Biomol. Chem., 2012, 10, 7840–7846
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Fig. 5 Emission spectra of A. The tethered donor–acceptor FRET peptides and B. FRET peptide after enzymatic cleavage, (DMSO 1 × 10−5 M),
λexc = 340 nm.
the formation of two fragments 4 and 5 that separate the fluoro-
phore from the quencher (Fig. 6).
Therefore, the fluorescence from the DM containing fragment
is recovered as the two pairs are no longer in the proximity
required for the intramolecular FRET to occur. The emission
profile of the FRET system before and after the enzymatic clea-
vage are presented on Fig. 5A and 5B and clearly indicate a
change in the fluorescence signal attributed to the efficient
energy transfer between the compatible donor–acceptor partners.
Diffusional quenching between the two FRET components in
solution was found to be negligible based upon titration experi-
ments in which the quencher was gradually introduced to a solu-
tion containing the donor. No significant quenching was
observed even at 1.5 equivalents of the quencher.†
A kinetic study of the enzymatic cleavage of the peptide
system revealed that the intensity of the restored fluorescence (in
RFU) is enhanced in a concentration-dependent fashion with a
Fig. 6 FRET peptide cleaved by β-secretase.
Km 0.29 μM and a Vmax 28.9 μM min−1.† These results
confirmed our expectation that the DM derivatives can be suc-
cessfully incorporated into FRET experiments.
fluorescence quenching energy acceptor, dimethylaminoazo-
benzene-2′-carboxylic acid (DABCYL) was attached at the
C-terminus (Fig. 6).
The donor emission which displays a maxima centered at λem
455 nm suitably overlaps with the absorption of the quencher
centered at λabs 440 nm, fulfilling one of the major requirements
for an efficient intramolecular FRET system.† The Förster-
Radius (R0) calculated for this FRET system is 36 Å.† The
proper quencher selection was confirmed in a preliminary experi-
ment in which the two molecules, 1b and the acceptor (Dabcyl)
were covalently linked through a coupling reaction (compound 9
in the ESI†) and the FRET-based quenching mechanism was
observed due to the close proximity between the FRET partners
(λex 340 nm). The absorption and the emission profile of 9 is
presented in the ESI† and clearly indicates that fluorescence
quenching occurs.† These results were consistent with the spec-
tral profile obtained from the actual FRET peptide system
(Fig. 5A and 5B). The FRET substrate entails a “fluorescence
dequenching” motif in which the quenched fluorophore partici-
pating in the intramolecular FRET becomes fluorescent once the
substrate/acceptor is cleaved by the enzyme, β-secretase. Incu-
bation of the FRET peptide with β-secretase (for 1 hour at room
temperature) results in the cleavage of the Leu–Asp bond and
FRET system with bright fluorophore moiety as an acceptor
Encouraged by these results, the application of 1 was expanded
by the design of a second FRET system using another chromo-
phore as an acceptor-free-base tetraphenylporphyrin (TPP).10
This molecule was chosen as a suitable acceptor due to the TPPs
high molar extinction coefficient ε(417) 470 300 M−1 cm−1 (in
THF), sufficient spectral overlap with the emission band of the
donor molecule centered at λabs 450 nm and to the large Stokes
shift of the donor. The low energy emission (λmax ∼640 nm) of
the TPP avoids any undesired overlap between the donor and the
acceptor emissions and the primary absorption band of TPP
(Soret band ∼416 nm) is red shifted relative to the absorption
band of 1 which also minimizes the partial absorption of the
excitation energy by the porphyrin (primary inner-filter effect).
The synthetic pathway to the final FRET complex was done as a
result of a spontaneous reaction between 1a and the pre-activated
succinimidyl ester of TPP 8 (Fig. 7).†
When compound 7 was excited at 270 nm or 340 nm, signifi-
cant fluorescence enhancement typical for the acceptor emission
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Conclusions
In summary, we have presented new fluorophore 3,4-bis(2,4-
difluorophenyl)-maleimide 1 and its derivatives that possess
favorable fluorescence properties suitable for biological and bio-
medical applications. In addition we demonstrated that 1 can
easily be decorated with functional handles that enhance its con-
jugation to the biological target of interest, without altering its
fluorescent properties. Furthermore, for the first time we demon-
strated the implementation of the DM moiety as an acceptor unit
in two FRET systems.
Experimental section
Fig. 7 Synthesis of the FRET paired system.
General information
All of the reagents and solvents are commercially available and
were used as purchased, without further purification. Column
chromatography was carried out using Merck Kieselgel 60 H
silicagel. 1H NMR, 13C NMR were recorded on a Bruker
250 MHz and Varian 400 MHz NMR spectrometer. All 1H
NMR experiments were reported in δ units, parts per million
(ppm) downfield of TMS and were measured relative to the
signals for chloroform (7.26 ppm) and deuterated methanol
(3.35, 4.78 ppm). All 13C NMR spectra were reported in ppm
relative to the signals for chloroform (77 ppm) and dimethylsulf-
oxide (49.3 ppm) with 1H decoupled observation. HRMS were
taken on an Agilent G1969A LC/MSD TOF. Fluorescence
measurements were conducted using a Photon Counting Spectro-
photometer ISS (PC1) and the absorption measurements were
recorded on Shimadzu UV-2401-PC. Absorption and fluor-
escence spectra were recorded in 1 cm path length quartz
cuvette. All solvents used for spectroscopy experiments were
spectrophotometric grade.
Fig. 8 Diffusional FRET between the non-covalently bound donor–
acceptor pair. Emission spectra were obtained by gradual titration of the
acceptor into a donor containing sample (λexc = 340 nm, 2.5 × 10−5
of 1a in toluene).
M
was observed (λmax ∼640 nm). This is particularly important
since in a control experiment it was confirmed that the acceptor
unit alone does not emit under these conditions.† The Förster-
Radius (R0) calculated for this FRET system is 26 Å. The FRET
efficiency was qualitatively estimated by quantifying the emis-
sion ratio change (RRC). This value was measured by taking the
product between the intensity of the donor and the acceptor’s
emissions at their respective maxima for the covalently attached
entity 7 and dividing that by the same product for an equimolar
sample of the free components in solution (see ref. 11 for
description of method). The experimental data revealed that
there was a 4.5-fold signal increase in the case of the covalently
linked complex† which is in a similar range as SFP/YFP
(RRC = 3), Cerulean/YFP (RRC = 2), and BFP/GFP (RRC = 5)
FRET pairs.11 In addition, diffusional FRET was explored in a
concentration dependent manner by two parallel titration experi-
ments in which one of the chromophores was kept at a constant
concentration, and the other was titrated, monitoring the emis-
sion signal after each titration step. In both experiments, regard-
less of which component was kept at a constant concentration,
an increase in the fluorescence intensity of the acceptor emission
and a simultaneous decrease in the emission from the donor
were observed due to diffusional quenching/FRET (Fig. 8).
3,4-Bis(2,4-difluorophenyl)-maleimide 1. A suspension of
(2,4-difluoro-phenyl)-acetonitrile (153 mg, 1 mmol) and iodine
(254 mg, 1 eq) in diethyl ether was reacted with freshly prepared
sodium methoxide in methanol solution (113 mg, 2.1 mmol in
10 mL dry methanol) at −85 °C. The reaction mixture was
brought to room temperature and after two hours it was treated
with 3% HCl until reaching pH 7. The reaction was completed
after stirring at room temperature for 24 hours and the crude
mixture was washed with water (20 mL), saturated solution of
sodium chloride (15 mL), and extracted with diethyl ether
(30 mL × 3). After extraction with diethyl ether, the combined
organic phases were dried over anhydrous sodium sulfate and
concentrated. The product 1 (177 mg, 55%) was obtained by
flash chromatography (DCM : methanol = 10 : 2). Rf = 0.55
1
(DCM : methanol = 9 : 2). H NMR (250 MHz, CDCl3) δ 8.44
(s, 1H), 7.50 (td, J = 8.3, 6.4 Hz, 2H), 6.97 (td, J = 8.0, 2.4 Hz,
2H), 6.85–6.77 (m, 2H). 13C NMR (63 MHz, CDCl3) δ
169.49 (s), 166.37–158.64 (m), 134.39 (s), 132.48 (dd, J = 9.9,
4.3), 113.34 (ddd, J = 15.2, 4.0), 112.10 (dd, J = 21.6, 3.6 Hz,
7H), 104.84 (t, J = 25.4 Hz). DEPT 90 13C NMR (63 MHz,
CDCl3) δ 132.37 (dd, J = 10.0, 4.3 Hz), 111.99 (dd, J = 21.6,
3.7 Hz), 104.74 (t, J = 25.4 Hz). HRMS (ESI) calcd for
C16H7F4NO2 [M + H]+: 322.0412, found: 322.0485.
7844 | Org. Biomol. Chem., 2012, 10, 7840–7846
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1-(2-Aminoethyl)-3,4-bis(2,4-difluorophenyl)-1H-pyrrole-2,5-
dione 1a. The synthesis of (2-bromo-ethyl)-carbamic acid tert-
butyl ester were performed according to a reported procedure.1
The suspension of 3,4-bis-(2,4-difluorophenyl)-maleimide 1
(321 mg, 1 mmol) in 10 mL dry DMF was cooled to 0 °C and
reacted with (2-bromo-ethyl)-carbamic acid tert-butyl ester
(336 mg, 1.5 eq) and potassium tert-butoxide (2 eq, 225 mg)
under inert atmosphere. The reaction was completed after
36 hours and the crude mixture was washed with saturated solu-
tion of sodium bicarbonate and extracted with ethyl acetate
(3 × 20 mL). After extraction with ethyl acetate the combined
organic phases were dried over anhydrous sodium sulfate and
concentrated. The protected crude {2-[3,4-bis-(2,4-difluoro-
phenyl)-2,5-dioxo-2,5-dihydro-pyrrol-1-yl]-ethyl}-carbamic acid
tert-butyl ester 2 was not purified but further reacted with 5%
TFA solution in DCM and stirred at room temperature for
3 hours in order to remove the Boc group. The crude mixture
was washed with water (20 mL) and extracted with DCM
(3 × 20 mL). The combined organic phases were dried over
anhydrous sodium sulfate and concentrated. The product
(323 mg, 89%) was obtained by chromatography (DCM :
temperature and the crude mixture was washed with saturated
solution of sodium chloride (15 mL) and extracted with ethyl
acetate (3 × 30 mL). After extraction with ethyl acetate the com-
bined organic phases were dried over anhydrous sodium sulfate
and concentrated. The desired product 3 was (357 mg, 82%) was
obtained by chromatography (hexane : ethyl acetate = 6 : 4). Rf =
0.45 (hexane : ethyl acetate = 7 : 3). 1H NMR (250 MHz,
CDCl3) δ 7.43 (dd, J = 8.2, 6.4 Hz, 2H), 6.94 (td, J = 8.0,
2.4 Hz, 2H), 6.78–6.65 (m, 2H), 4.14–3.95 (m, 2H), 3.65 (t, J =
6.7 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H), 2.04–1.86 (m, 2H), 1.15
(dd, J = 10.4, 3.8 Hz, 3H). 13C NMR (63 MHz, CDCl3)
δ 172.63 (s), 169.47 (s), 162.40–158.51 (m), 133.29 (s), 132.48
(dd, J = 10.0, 4.2 Hz), 113.62 (ddd, J = 15.0, 3.8, 2.2 Hz),
111.91 (dd, J = 21.6, 3.6 Hz), 104.65 (t, J = 25.5 Hz), 60.59 (s),
38.08 (s), 31.64 (s), 23.87 (s), 14.21 (s). HRMS (ESI) calcd for
C22H17F4NO4, [M + H]+: 436.1094, found: 436.1078.
4-[3,4-Bis-(2,4-difluoro-phenyl)-2,5-dioxo-2,5-dihydro-pyrrol-
1-yl]-butyric acid 1c. The suspension of 4-[3,4-bis-(2,4-
difluoro-phenyl)-2,5-dioxo-2,5-dihydro-pyrrol-1-yl]-butyric acid
ethyl ester 3 (435 mg, 1 mmol) in 15 mL HCl : AcOH = 1 : 1
was refluxed at 90 °C for 20 hours. The crude product was
extracted with DCM (20 mL × 3) and the combined organic
phases were dried over anhydrous sodium sulfate and concen-
trated. The desired product 1c (326 mg, 80%) was obtained by
chromatography (DCM : methanol = 7 : 3). Rf = 0.52 (DCM :
1
methanol = 7 : 3). Rf = 0.55 (DCM : methanol = 8 : 2). H NMR
(400 MHz, CD3OD) δ 7.57 (td, J = 8.4, 6.4 Hz, 2H), 7.09–7.03
(m, 2H), 7.03–6.95 (m, 2H), 3.99–3.94 (m, 2H), 3.27–3.23 (m,
2H), 1.26 (s, 2H). 13C NMR (63 MHz, CDCl3) δ 169.55 (s),
166.22–158.62 (m), 133.29 (s), 132.47 (dd, J = 9.9, 4.4 Hz),
113.67 (ddd, J = 15.1, 3.9, 2.2 Hz), 111.94 (dd, J = 21.6,
3.6 Hz), 104.70 (t, J = 25.6 Hz), 38.31 (s), 18.03 (s). HRMS
(ESI) calcd for C18H12F4N2O2 [M + H]+: 365.0835, found:
365.0896.
1
methanol = 8 : 2). H NMR (250 MHz, CDCl3) δ 7.43 (td, J =
8.3, 6.5 Hz, 2H), 6.97 (td, J = 8.0, 2.4 Hz, 2H), 6.80–6.69 (m,
2H), 3.68 (t, J = 6.8 Hz, 2H), 2.39 (t, J = 7.3 Hz, 2H), 1.98 (dd,
J = 14.2, 7.2 Hz, 2H). 13C NMR (63 MHz, CDCl3) δ 178.91 (s),
169.60 (s), 167.25–156.95 (m, 2H), 133.38 (s), 132.50 (dd, J =
10.0, 4.1 Hz), 113.58 (ddd, J = 15.3, 3.9, 2.2 Hz), 112.03 (dd,
J = 21.6, 3.5 Hz), 104.77 (t, J = 25.6 Hz), 38.01 (s), 31.40 (s),
23.49 (s). HRMS (ESI) calcd for C20H13F3NO4, [M + H]+:
408.0781, found: 408.0742.
3,4-Bis(2,4-difluorophenyl)-1-(3-hydroxypropyl)-1H-pyrrole-
2,5-dione 1b. The suspension of 3,4-bis(2,4-difluorophenyl)-
maleimide 1 (321 mg, 1 mmol) in dry 10 mL DMF was cooled
to 0 °C and was reacted with potassium tert-butoxide (2 eq,
225 mg) and 3-bromopropan-1-ol (1.5 eq, 207 mg) under inert
atmosphere. The reaction was completed after 24 hours at room
temperature and the crude mixture was washed with saturated
solution of sodium bicarbonate (15 mL) and extracted with
DCM (3 × 30 mL). After extraction with ethyl acetate the com-
bined organic phases were dried over anhydrous sodium sulfate
and concentrated. The desired product 1b (322 mg, 85%) was
obtained by chromatography (DCM : methanol = 10 : 3). Rf =
N-{2-[3,4-Bis-(2,4-difluoro-phenyl)-2,5-dioxo-2,5-dihydro-pyrrol-
1-yl]-ethyl}-4-(10,15,20-triphenyl-porphyrin-5-yl)-benzamide 7.
The synthesis of 4-(10,15,20-triphenyl-porphyrin-5-yl)-benzoic
acid 2,5-dioxo-pyrrolidin-1-yl ester 8 was performed by follow-
ing a reported procedure2 and the precursor of 8, 4-(10,15,20-
triphenyl-porphyrin-5-yl)-benzoic acid was obtained through
hydrolysis following a previously reported procedure.3 The
suspension of 4-(10,15,20-triphenyl-porphyrin-5-yl)-benzoic
acid 2,5-dioxo-pyrrolidin-1-yl ester 8 (755 mg, 1 mmol) in
10 mL dry DCM was reacted with 1-(2-amino-ethyl)-3,4-bis-
(2,4-difluoro-phenyl)-pyrrole-2,5-dione (1a) (364 mg, 1 eq). The
reaction mixture was stirred for 24 hours at room temperature
and the product formation was monitored by TLC and MALDI-
TOF. The desired product 7 (904 mg, 90%) was obtained by
chromatography (DCM : methanol = 9 : 1). Rf = 0.50 (100%
DCM). MALDI-TOF, calculated for: 1006.309, found:
1
0.45 (DCM : methanol = 8 : 2). H NMR (250 MHz, CDCl3) δ
7.49 (td, J = 8.3, 6.4 Hz, 2H), 6.96 (ddd, J = 8.0, 2.5, 1.3 Hz,
2H), 6.86–6.74 (m, 2H), 3.82 (t, J = 6.4 Hz, 2H), 3.66 (t, J =
5.7 Hz, 2H), 1.93–1.82 (m, 2H). 13C NMR (63 MHz, CDCl3
δ 169.93 (s), 166.22–158.57 (m), 133.39 (s), 132.41 (dd, J =
9.9, 4.3 Hz), 113.33 (ddd, J = 15.2, 4.0), 111.94 (dd, J = 21.6,
3.6 Hz), 104.69 (t, J = 25.4 Hz), 59.33 (s), 35.36 (s), 31.33 (s).
HRMS (ESI) calcd for C19H13F4NO3, [M + H]+: 380.0832,
found: 380.0812.
1
1006.310. H NMR (250 MHz, CDCl3) δ 8.89 (d, J = 5.5 Hz,
4-(3,4-Bis-(2,4-difluorophenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-
1-yl)butanoate 3. The suspension of 3,4-bis(2,4-difluorophenyl)-
maleimide 1 (321 mg, 1 mmol) in 10 mL dry DMF was cooled
to 0 °C and was reacted with potassium tert-butoxide (2 eq,
225 mg) and with 4-bromo-butyric acid (293 mg, 1.5 eq) under
inert atmosphere. The reaction was stirred for 24 hours at room
5H), 8.81 (d, J = 4.8 Hz, 2H), 8.46 (d, J = 8.2 Hz, 2H), 8.33 (d,
J = 8.1 Hz, 2H), 8.29–8.18 (m, 6H), 7.77 (td, J = 4.7, 2.3 Hz,
9H), 7.51 (td, J = 8.3, 6.5 Hz, 2H), 6.99 (td, J = 8.2, 2.1 Hz,
2H), 6.82 (ddd, J = 11.0, 8.9, 2.4 Hz, 2H), 3.78 (t, 2H), 2.46
(t, J = 7.3 Hz, 2H), −2.73 (s, 1H). 13C NMR (63 MHz, CDCl3)
δ 169.99, 169.24, 167.47–158.54 (m), 147.16 (s), 142.12 (s),
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134.64 (s), 133.40 (s) 132.44 (dd, J = 11.0, 5.1 Hz), 129.62 (s),
129.55 (s) 128.05 (s), 128.87 (s) 126.81 (s), 120.70 (s), 120.51
(s), 118.68 (s), 113.55 (m), 112.01 (dd, J = 21.6, 3.6 Hz),
104.77 (t, J = 25.6 Hz), 52.52 (s,), 31.33 (s). MALDI-TOF,
calculated 1004.31, found 1005.33.
Notes and references
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4-(4-Dimethylamino-phenylazo)-benzoic acid 3-[3,4-bis-(2,4-
difluoro-phenyl)-2,5-dioxo-2,5-dihydro-pyrrol-1-yl]-propyl ester
9. A suspension of 1b, 3,4-bis-(2,4-difluoro-phenyl)-1-prop-2-
ynyl-pyrrole-2,5-dione, (379 mg, 1 mmol) in dry 10 mL DCM
was reacted with 1-ethyl-3-(3′-dimethylaminopropyl) carbodi-
imide (287 mg, 1.5 eq), 4-dimethylaminopyridine (61 mg,
0.5 eq) and 4-(4-dimethylamino-phenylazo)-benzoic acid
(DABCYL) (269 mg, 1 eq). The reaction mixture was stirred for
24 hours at room temperature and the product formation was
monitored by TLC and LC/MS. The cruder material was washed
with water and extracted with DCM (20 mL × 3). The combined
organic phases were dried over anhydrous sodium sulfate and
concentrated. The desired product 9 (560 mg, 80%) was obtained
by chromatography (DCM : methanol = 8 : 2). Rf = 0.52 (DCM :
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methanol = 8 : 2). H NMR (250 MHz, CDCl3) δ 8.13 (d, J =
8.6 Hz, 2H), 7.86 (dd, J = 18.7, 8.8 Hz, 4H), 7.44 (td, J = 8.2,
6.5 Hz, 2H), 6.75 (td, J = 8.0, 2.4 Hz, 2H), 6.77 (ddd, J = 9.2,
3.5, 1.8 Hz, 4H), 4.43 (t, J = 5.9 Hz, 2H), 3.90 (t, J = 6.7 Hz,
2H), 2.30–2.14 (m, 2H). 13C NMR (63 MHz, CDCl3)
δ 169.47 (s), 166.53 (s), 162.46–156.14 (m), 153.02 (s),
143.81 (s), 133.40 (s), 132.49 (dd, J = 10.0, 4.1 Hz), 130.62 (s),
130.06 (s), 125.63 (s), 122.14 (s), 113.77 (ddd, J = 15.1, 3.9,
2.2 Hz), 112.19 (dd, J = 21.6, 3.6 Hz), 104.74 (t, J = 25.6 Hz),
104.34 (s), 62.70 (s), 40.38 (s), 36.29 (s), 27.81. HRMS (ESI)
calcd for C34H26F4N4O4, [M + H]+: 631.1890, found: 631.1855.
7846 | Org. Biomol. Chem., 2012, 10, 7840–7846
This journal is © The Royal Society of Chemistry 2012