Fluorescent triphenyl substituted maleimide derivatives: Synthesis, spectroscopy and quantum chemical calculations

Article abstract of DOI:10.1007/s10895-010-0660-y

In this paper we describe a semi-empirical quantum method for predicting the wavelength of maximum fluorescence excitation and emission for several known and new maleimide derivatives. All new maleimides, containing a N-Benzyl attachment, were successfully synthesised via a tandem Suzuki reaction with aryl boronic acids containing either an electron donating, electron withdrawing functional groups. Absorption and emission spectra calculated using the semi-empirical AM1 method with excited state ZINDO calculations proved more reliable than either Hartree-Fock Configuration interaction or time dependent density functional methods. Calculated absorption and emission wavelengths were compared with 26 experimental spectra from known or newly synthesised maleimides and found to have provide reasonable predictions, with an average deviation of less the 6% for absorption maxima and less than 4% for emission peaks. The described method provides a strong benchmark for the accuracy that can be expected from theoretical predictions of fluorescence spectra. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s10895-010-0660-y

J Fluoresc (2010) 20:1077–1085
DOI 10.1007/s10895-010-0660-y
ORIGINAL PAPER
Fluorescent Triphenyl Substituted Maleimide
Derivatives: Synthesis, Spectroscopy and Quantum
Chemical Calculations
Hui-ding Xie & Louisa A. Ho & Michael S. Truelove &
Ben Corry & Scott G. Stewart
Received: 3 November 2009 /Accepted: 29 March 2010 /Published online: 15 April 2010
#
Springer Science+Business Media, LLC 2010
Abstract In this paper we describe a semi-empirical
quantum method for predicting the wavelength of maxi-
mum fluorescence excitation and emission for several
known and new maleimide derivatives. All new malei-
mides, containing a N-Benzyl attachment, were successful-
ly synthesised via a tandem Suzuki reaction with aryl
boronic acids containing either an electron donating,
electron withdrawing functional groups. Absorption and
emission spectra calculated using the semi-empirical AM1
method with excited state ZINDO calculations proved more
reliable than either Hartree-Fock Configuration interaction
or time dependent density functional methods. Calculated
absorption and emission wavelengths were compared with
26 experimental spectra from known or newly synthesised
maleimides and found to have provide reasonable predic-
tions, with an average deviation of less the 6% for
absorption maxima and less than 4% for emission peaks.
The described method provides a strong benchmark for the
accuracy that can be expected from theoretical predictions
of fluorescence spectra.
Introduction
The design and synthesis of new fluorescent molecules is of
continuing interest for both understanding the fundamental
electronic structure of molecules as well as for many
applications in research and industry. Fluorescent dyes have
found an enormous number of applications in chemical and
biological research, where they can be used for fluorometric
detection in high performance liquid chromatography [1, 2],
in microscopy and spectrometry to isolate the location of
organelles or proteins [3, 4], detect electrical or chemical
changes in the environment [5] or to isolate protein
interactions or conformational changes [6]. To this end,
fluorophores linked to unsubstituted maleimides are often
used as thiol reactive probes [7, 8]. Substituted maleimides
which have extended conjugation compounds, sometimes
naturally occurring, which recently have been shown to
inhibit protein kinase C [9].
The ability to design new fluorescent molecules in silico
before chemical synthesis can expedite the process of
constructing dyes with desired properties. Although the
effect of the chemical structure on the fluorescent character-
istics of the molecules has been studied numerous times
[10–25] there is no simple strategy available for accurately
predicting these characteristics directly from the molecular
structure. From an experimental perspective, reported
trends suggest adding electron donating groups to aromatic
compounds increases the absorption maximum is as
expected. More specifically, reports indicate the addition
of a dimethyl amino group –N(CH3)2 to an a aromatic ring
increases the wavelength of the maximum in fluorescence
intensity, while groups such as methoxy and hydroxyl have
more moderate influences. For fluorescence emission
spectra it is regularly reported that the addition of an
electron donating group increases the wavelength of
.
.
Keywords Maleimide Quantum chemistry
.
.
Semi-empirical calculation Fluorescence Synthesis
H.-d. Xie
Department of Chemistry, Kunming Medical College,
Kunming 650031, China
:
:
:
L. A. Ho M. S. Truelove B. Corry (*) S. G. Stewart
School of Biomedical, Biomolecular and Chemical Sciences,
The University of Western Australia,
Crawley 6009, Australia
e-mail: ben.corry@uwa.edu.au

1078
J Fluoresc (2010) 20:1077–1085
Scheme 1 Reagents and
conditions: a BnNH2, AcOH,
50 °C, 16 h, 81%; b PdCl2(dppf)
(10 mol%), NaOH boronic acid,
rt, or Pd2(dba)3.CHCl3, dppf,
NaOH, MeNCy2 and boronic
acid, rt, 6–45%
emission. Other studies report that adding electron with-
drawing groups, and thus normally conjugation, has a
stronger influence on the fluorescence quantum yield,
however differences between the type of electron with-
drawing functional groups and the excitation and emission
maximum varies remarkably [13–15].
excellent yield (81%). Subjecting this compound to a
tandem Suzuki cross coupling reaction afforded the bis-
aryl compound in a range of yields, depending on the para-
substituted R group. The two catalytic conditions, deemed
most reliable in the tandem reaction, were PdCl2(dppf),
NaOH, phenylboronic acid (2.5 equiv) and a method
applying an in situ formation of Pd(dppf) through
Pd2(dba)3. Although it was assumed that yields could have
been improved by changing the substrate 2 to the diiodo
precursor it was not considered necessary for this physical
characteristic study [29–31]. The resulting arylated com-
pounds were purified under standard chromatography
conditions (Scheme 1).
As excitation and emission spectra are determined by the
electronic energy levels of the molecules, the prediction of
accurate fluorescent properties requires accurate electronic
structures to be determined. To avoid prohibitive computa-
tional demands, such calculations are often done with
Hartree–Fock theory (HF), time dependent density func-
tional theory (TDDFT) or semiempirical methods [17]. It is
still not clear, however, how accurately each of these
approaches can reproduce observed spectra. Semiempirical
approaches are the least time consuming and have recently
given promising results for maleimide derivatives [18, 19]
in addition to many other molecules including porphyrins
[20] and benzofurazans. However, recent improvements in
computational power has meant that other approaches are
feasible, and TDDFT [21–24] and ab-initio configuration
interaction [25] approaches have had success in some cases.
To help understand the limitations of the methods, we
calculate spectra for well characterized AlexaFluor dyes, as
well as a maleimide derivative with each of these
approaches. We also test the accuracy of less computation-
ally demanding semiempirical AM1/ZINDO approach
across all six new maleimide derivatives as well as nineteen
previously synthesized molecules [26] to gain an appreci-
ation of the uncertainty in such calculations.
Calculations
Unless stated elsewhere, absorption and emission spectra
were calculated using semi-empirical quantum chemical
methods with HyperChem7.5. To calculate absorption
spectra, the molecules were first geometrically optimised
using the semi-empirical AM1 method [32] (RMS gradient
was set to 0.01). Once this was done, a single point excited
state calculation was made using the ground state geometry
using the ZINDO/S method [33, 34]. To calculate the
emission spectrum the molecules geometry optimisation
was made in the excited state using the ZINDO/l method
[35] (RMS gradient was set to 0.1), with the aim of
simulating internal conversion without any emission. After
the optimization was completed, the semi-empirical method
was set back to ZINDO/S for single-point energy calcula-
tion. An emission spectrum was generated when the single-
point energy calculation was completed.
Methods
Table 1 Comparison of calculation methods. Wavelength of maxi-
mum absorbance calculated by three different methods for three
compounds are shown along with the associated experimental value
Synthesis
The synthesis of symmetric bis-aryl maleimides has been
explored by several synthetic groups including ours [26,
27]. Our synthetic plan was devised on previous work
within this group and others involving the cross coupling of
halogenated phthalimides [28–30]. Treatment of the com-
mercially available 2,3-dichloromaleic anhydride (1) with
benzylamine furnished the protected maleimide 2 in
AM1-ZINDO HF-CIS
TDDFT
Experimental
data
4
374.1 (0.479) 255.1(0.429) 371.0(0.141)
434.9 (0.530) 265.8(0.451) 404.5(0.059)
350
448
493
23
AF488 444.4(0.651)
319
666

J Fluoresc (2010) 20:1077–1085
1079
For comparison, some additional calculations were made
using Hartree–Fock Configuration interaction (singles) (HF-
CIS) [36] and time dependent density functional theory (TD-
DFT) using the B3LYP functional as described in the results.
Similar results were obtained using a range of basis sets,
however, all results shown employ 6–31G*. All ab initio and
DFT calculations were made using Gaussian03 [37].
Experimentally, the fluorescence spectra were all determined
in hexane, except for compound 23 where dichloromethane
was used for solubility purposes. A consistent choice of
solvent is paramount considering reports that fluorescence is
highly dependent on solvent polarity. A low solvent with low
dielectric constant was chosen for the best comparison with
gas phase theoretical calculations.
Fig. 1 Structures of reported
maleimide derivatives [25]

1080
J Fluoresc (2010) 20:1077–1085
Results
In general, the increase in the wavelength of the
excitation and emission maxima with the addition of
electron donating groups such as -OMe and -NR2 is
shown, as highlighted in the differences in compound 4 in
comparison to derivatives 14 and 22. Similarly, the
addition of heteroatoms (compounds 16 and 17) and
additional conjugation (compounds 6 and 7) has the same
effect in the system. In the last instance electron
withdrawing groups which do not extend the conjugated
system (i.e. compound 13) have the opposite effect to their
electron donating counterparts and decrease both excita-
tion and emission wavelengths. In each of these cases the
theoretical calculations have been able to predict these
changes accurately [38, 39].
The results, presented graphically in 2, highlight the
broad level of consistency obtained between the calculated
and experimentally determined values. An obvious trend is
apparent in the predicted excitation peaks, with the wave-
lengths being overestimated for compounds absorbing at
short wavelengths and underestimated for those absorbing
at long wavelengths. While we are not clear as to the
origins of this trend, it may be related to the parameter-
isation of the semiempirical calculations performing less
well for molecules with non-local valence electrons. A
similar level of agreement is seen in the emission wave-
lengths as for excitation, but no systematic differences with
the experimental data is seen.
Assessing calculation accuracy
To determine the most appropriate method to use for
calculating the fluorescent properties of the compounds, we
calculated the absorbance properties of two of the maleimide
derivatives and one well known organic dye using semi-
empirical AM1, HF-CIS and TDDFT methods as shown in
Table 1. In all cases, the wavelength of maximum
absorption determined using the HF-CIS method was found
to be well below the expected value. Altering the basis set
(up to 6–311++G**) had little influence on the results
suggesting a systematic failure of the approach. Results
from TDDFT were more varied, but proved extremely poor
for the Alexafluor dye. Again, altering the basis set had
little effect. Surprisingly, results from TD-HF theory were
much better for AF488 (345 nm) than the equivalent
B3LYP result. Semi-empirical calculations appeared more
reliable and were utilised for the remainder of the
calculations described.
To gain an appreciation of the reliability of the semi-
empirical calculations, absorption and emission wavelengths
were determined for 19 maleimide derivatives (Fig. 1) that
have been previously characterised as shown in Table 2
arranged according to increasing wavelength of absorption
(Fig. 2).
Table 2 Calculated (in vacuo) and experimental data [25] (in hexane) wavelengths of maximum absorption and emission of known maleimide
derivatives. Calculated oscillator strengths are shown in parentheses
abs
max
abs
em
max
em
l
ðnmÞ calculated
l
ðnmÞ
l
ðnmÞ calculated
l
ðnmÞ
Compound
Dev. (%)
Dev. (%)
max
max
experimental
experimental
4
374.1 (0.479)
377.5 (0.413)
361.9 (0.290)
360.4 (0.288)
400.1 (0.573)
402.2 (0.550)
361.2 (0.515)
364.9 (0.388)
361.4 (0.519)
366.4 (0.408)
396.6 (0.515)
397.8 (0.479)
375.8 (0.572)
449.2 (0.896)
448.7 (0.798)
428.2 (0.614)
441.0 (0.560)
437.7 (0.585)
418.6 (0.507)
350
361
373
377
391
395
339
352
339
350
387
399
340
414
423
474
476
480
481
6.89
4.57
473.1 (0.457)
482.2 (0.314)
465.4 (0.251)
460.5 (0.268)
525.4 (0.612)
509.7 (0.562)
447.4 (0.480)
455.1 (0.312)
449.2 (0.484)
464.4 (0.322)
507.3 (0.523)
512.0 (0.421)
461.1 (0.532)
526.2 (0.939)
548.1 (0.652)
596.0 (0.670)
581.7 (0.620)
580.4 (0.690)
553.7 (0.550)
471
490
497
507
488
499
460
477
459
475
506
518
464
514
530
572
575
581
581
0.45
−1.59
−6.36
−9.17
7.66
5
6
−2.98
−4.40
2.33
7
8
9
1.82
2.14
10
11
12
13
14
15
16
17
18
19
20
21
22
6.55
−2.74
−4.59
−2.14
−2.23
0.26
3.66
6.61
4.69
2.48
−0.30
10.53
8.50
−1.16
−0.63
2.37
6.08
3.42
−9.66
−7.35
−8.81
−12.97
4.20
1.17
−0.10
−4.70

J Fluoresc (2010) 20:1077–1085
1081
480
460
440
420
400
380
360
340
320
deviation in the wavelength of maximum emission (−3.9
and −6.9% respectively). Again a trend is apparent in the
predicted excitation peaks, with the wavelengths being
overestimated for compounds absorbing at short wave-
lengths (c.a 340–370 λ max i.e. compounds 25 and 27) and
underestimated for those absorbing at long wavelengths (i.e.
compound 23 and 24, 27 and 28). However, in the
fluorescence emission series the calculated values tended to
be under estimated in each of the N-benzylated cases.
Conclusions
320
340
360
380
400
420
440
460
480
Experimental Absorption Maximum (nm)
In summary, this paper has compared experimental fluores-
cence data from known and new maleimide compounds
with predicted data. Semi-empirical quantum AM1 method
with excited state ZINDO calculations were used for
predicting the wavelength of the maximum fluorescence
excitation and emission. Calculated absorption and emis-
sion wavelengths were within an average deviation of less
the 6% for absorption maxima and less than 4% for
emission peaks. All new maleimides were successfully
synthesised via a tandem Suzuki reaction. We expect the
described method considering its accuracy can be used for
future theoretical predictions of fluorescence spectra.
600
580
560
540
520
500
480
460
440
Experimental
440
460
480
500
520
540
560
580
600
Experimental Emission Maximum (nm)
Nuclear Magnetic Resonance (NMR) spectra were recorded
on a Bruker ARX 300 (1H at 300.14 MHz and 13C at
75.47 MHz), Bruker AV 500 (1H at 500.13 MHz and 13C
at 125.8 MHz), Varian 400 (1H at 399.86 and 13C at
100.54 MHz) or a Bruker AV 600 (1H at 600.13 MHz and
Fig. 2 Tests of calculation accuracy. Calculated wavelengths of
maximum excitation (a) and emission (b) are plotted against
experimental data for the six new compounds described here as well
as the 19 previously reported maleimide derivatives
1
13C at 150.90 MHz) spectrometer at 25 °C. H and 13C
Excitation and emission spectra of new compounds
spectra were referenced to the residual (partially) undeuter-
ated solvents. Infrared (IR) spectra were recorded with a
PerkinElmer Spectrum One Spectrometer FT-IR spectrome-
ter. Samples were analysed as thin films on NaCl discs. Mass
Spectra were collected using electron impact ionisation (EI-
MS) on a VG AutoSpec. EI-HRMS was performed with a
resolution of approximately 10,000. All air and/or moisture
sensitive reactions were performed in flame dried glassware
under an argon atmosphere. All solvents were distilled prior
to use, and if used in air and/or moisture sensitive reactions,
were degassed. Anhydrous solvents were obtained by
distillation from the appropriate drying agent. Thin layer
chromatography (TLC) was performed on Merck silica gel
60 F254, pre-coated aluminium sheets. Visualisation of
developed plates was achieved through the use of a
254 nm or 365 nm UV lamp. Column chromatography was
performed using silica gel 60 (0.063–0.200 mm) as supplied
by Merck with the eluents indicated. Starting materials and
In the newly synthesised N-Benzyl maleimides (23–28)
Fig. 3, the inclusion of electron donating substituents such
as -NMe2, -OiPr, -OH (compound 24, 27 and 28 respec-
tively), greatly increases the wavelength of both the
absorbance and emission maximum spectra experimentally
when compared to 25 (see Fig. 4 and Table 3), however, the
inclusion of the carbonyl moiety has little effect. Such shifts
in the wavelengths of the maxima are also observed in work
by Hirano on electronic effects in coelenteramide ana-
logues. In most cases these effects are predicted in the semi-
empirical quantum calculations with reasonable accuracy,
however, the error associated with the absorbance spectra of
compound 24 is larger. In the case of the thiophene
heterocyclic attachment (compound 23) and the electron
withdrawing acyl derivative (compound 27) the experimen-
tal and calculated values are similar with a slightly larger

1082
J Fluoresc (2010) 20:1077–1085
Fig. 3 Structures of new
maleimide derivatives (23–28)
reagents were generally available from Sigma–Aldrich or
Fluka.
in THF (5 mL) was degassed using the freeze pump thaw
method. The mixture was heated to 45 °C and left to stir for
16 h. The crude mixture was fused to silica and passed
through a plug of coarse silica with 1:1 ethyl acetate/
hexane. The filtrate was again fused to silica and subjected
to column chromatography eluting with (1:9 → 1:3 ethyl
acetate/hexane) to afford the bis-aniline 22 (42 mg, 6%) as
a deep red solid, m.p 191–193 °C; Rf=0.24 (3:7 ethyl
acetate/hexane); 1H NMR (400 MHz, CDCl3): δ=7.45–7.38
(m, 4H), 7.38–7.36 (m, 2H), 7.26–7.18 (m, 4H), 6.57 (d, J=
8.8 Hz, 4H, Ar–H), 4.69 (s, 2H, CH2), 2.92 (s, 12H,
4× CH3N); 13C NMR (400 MHz, CDCl3): δ=171.8 (C=O),
137.2, 131.2, 128.8, 128.7, 127.7, 111.8, 41.7 (CH2), 40.3
1-Benzyl-3,4-di-thiophen-2-yl-pyrrole-2,5-dione (23) 1,1′-
Bis(diphenylphosphino)ferrocene, (134 mg, 0.24 mmol)
was added in one portion to a magnetically stirred solution
of Pd(OAc)2 (56 mg, 0.24 mmol) in dry THF (3 mL). The
ensuing mixture was stirred for approximately 30 min
before being treated with 3,4-dichloromaleimide 2 (281 mg,
1.1 mmol) in one portion and stirred at room temperature
for 15 min. 4,4,5,5-Tetramethyl-2-thiophen-2-yl-[1,3,2]
dioxaborolane (500 mg, 2.4 mmol) and NaOH (97,
2.4 mmol) were then added in one portion to the reaction
mixture. The ensuing solution was then stirred at 46 °C for
16 h. Subjection of the crude material to flash column
chromatography (1:19 EtOAc/hexane) afforded the disub-
situted maleimide 20 (116 mg, 30%) as an orange solid.
M.p 130–132 °C; 1H NMR (300 MHz, CDCl3): δ=7.82 (dd,
J=1.2, 3.9 Hz, 2H, ArH), 7.57 (dd, J=1.2, 5.1 Hz, 2H, ArH),
7.46–7.42 (m, 2H, ArH), 7.37–7.28 (m, 3H, ArH), 7.12 (dd,
J=3.6, 5.1 Hz, 2H, ArH), 4.79 (s, 2H); 13C NMR
(100.5 MHz, CDCl3): 170.0 (C=O), 136.3, 131.5, 131.1,
129.8, 128.9, 128.8, 128.1, 127.7, 42.3 (CH2Ph); IR (neat)
υmax (cm−1): 2,925, 1,765, 1,700 (C=O), 1,432, 1,401, 699;
EI-MS m/z (relative intensity): 351 (100, [M]+), 323 (13),
246 (12), 191 (15), 190 (38); EI-HRMS calcd for
C19H13NO2S2: 351.0388, found: 351.0386.
~
(N(CH3)2); IR (film): n ¼ 2; 895 cmꢀ1, 1,695, 1,604, 1,354,
1,196, 818; MS (EI): m/z (%)=425.1 (100, [M]+); EI-
HRMS calcd for C27H27N3O2 425.2103; found: 425.2107.
1-Benzyl-3,4-diphenyl-1H-pyrrole-2,5-dione (25) 3,4-
Dichloromaleimide 2 (400 mg, 1.56 mmol), PdCl2(dppf).
CH2Cl2 (127 mg, 0.16 mmol), NaOH (125 mg, 3.1 mmol),
phenylboronic acid (415 mg, 3.4 mmol) in THF (5 mL) was
degassed using the freeze pump thaw method. The ensuing
mixture was stirred at room temperature for 16 h. The crude
mixture was fused to silica and subjected to column
chromatography, silica gel eluted with (1:19 → 1:4 ethyl
acetate/hexane), to afford the bis-aryl compound 23 as a
light green solid (243 mg, 46%), Rf=0.29 in 1:9 ethyl
acetate/hexane), m.p 132–134 °C. 1H NMR (300 MHz,
CDCl3): δ=7.47–7.44 (m, 6H, Ar–H), 7.36–7.30 (m, 9H,
Ar–H), 4.80 (s, 2H, N–CH2); 13C NMR (400 MHz,
CDCl3): δ = 170.6 (2C, 2 × C=O), 136.6, 136.3,
130.0,130.0, 129.0, 128.8, 128.7, 128.7, 128.1, 42.2
1-benzyl-3,4-bis(4-(dimethylamino)phenyl)-1H-pyrrole-2,5-
dione (24) 3,4-Dichloromaleimide 2 (400 mg, 1.6 mmol),
PdCl2(dppf).CH2Cl2 (127 mg, 0.16 mmol), 4-(N,N-dime-
thylamino)phenyl boronic acid (pinacol ester, 964 mg,
3.9 mmol), NaOH (187 mg, 4.7 mmol, in 0.5 mL water)
~
(CH2); IR (film): n ¼ 3; 060 cmꢀ1, 1,701 (C=O), 1,433,

J Fluoresc (2010) 20:1077–1085
1083
a
Pd2(dba)3.CHCl3 (161 mg, 0.16 mmol), dppf (173 mg,
0.16 mmol), NaOH (62 mg, 1.6 mmol), N-methyldicyclo-
hexylamine (334 µL, 1.6 mmol), 4-isopropoxyphenylboronic
acid (617 mg, 3.4 mmol) in THF (5 mL) was degassed using
the freeze pump thaw method. The ensuing mixture was
stirred at room temperature for 16 h, fused to silica and
passed through a plug of coarse silica with 1:1 ethyl acetate/
hexane. The concentrated organic washings were subjected
to column chromatography, 1:49 ethyl acetate/hexane to
100
80
60
40
23
1
24
25
afford the bis-ether 24 (69 mg, 9%) as an orange oil. H
NMR (300 MHz, CDCl3): δ=7.52–7.40 (m, 6H, Ar–H),
7.39–7.24 (m, 3H, Ar–H), 6.86 (m, 4H, Ar–H), 4.81 (s, 2H,
NCH2), 4.60 (septet, J=6.0 Hz, 2H, 2×CH), 1.37 (d, J=
6.0 Hz, 12H, 2×CH3), 13C NMR (300 MHz, CDCl3): δ=
171.2, 159.3, 136.8, 134.0, 131.6, 128.9, 128.8, 127.8,
121.1, 115.6, 70.0 (2×CH), 42.0 (CH2), 22.1 (2×CH3); IR
26
27
28
20
0
200
300
400
500
600
Excitation Wavelength (nm)
b
~
(film): n ¼ 3; 019 cmꢀ1, 2,980, 1,700 (C=O), 1,251, 1,215,
100
668; MS (EI): m/z (%)=455.1 (91, [M]+), 371.0 (100), 209.1
(25); EI-HRMS calcd for C29H29NO4 455.2097; found:
455.2109.
80
60
40
20
0
3,4-Bis(4-acetylphenyl)-1-benzyl-1H-pyrrole-2,5-dione
(27) 3,4-Dichloromaleimide 2 (400 mg, 1.6 mmol)
Pd2(dba)3.CHCl3 (161 mg, 0.16 mmol), dppf (173 mg,
0.16 mmol), NaOH (62 mg, 1.6 mmol), N-methyldicyclo-
hexylamine (334 µL, 1.6 mmol), 4-acetylphenylboronic
acid (562 mg, 3.4 mmol) in THF (5 mL) was degassed
using the freeze pump thaw method. The ensuing mixture
was left to stir at room temperature for 16 h. The crude
mixture was fused to silica and subjected to column
chromatography, silica gel eluted with (1:19 → 1:4 ethyl
acetate/hexane) to afford the bis-aryl compound 25 as a
light green solid (295 mg, 45%), Rf=0.30 in (2:3, ethyl
acetate/hexane), m.p 149–150 °C. 1H NMR (400 MHz,
CDCl3): δ=7.92–7.53 (m, 4H, Ar–H), 7.54–7.51 (m, 4H,
Ar–H), 7.45–7.41 (m, 2H, Ar–H), 7.32–7.29 (m, 3H, Ar–
H), 4.82 (s, 2H, N–CH2), 2.60 (s, 6H, 2×CH3); 13C NMR
(400 MHz, CDCl3): δ=197.4, 169.6, 138.0, 136.52, 136.12,
132.8, 130.2, 129.0, 128.9, 128.58, 128.18, 42.4, 26.8; IR
23
24
25
26
27
28
400
450
500
550
600
650
700
Emission Wavelength (nm)
Fig. 4 Measured excitation (a) and emission (b) spectra of new
maleimide derivatives
1,401, 1,350, 1,075, 692; MS (EI): m/z (%)=339.1 (100)
[M]+, 294.1 (32), 262.1 (21), 178.0 (37); EI-HRMS calcd
for C23H17NO2 339.1259; found: 339.1252.
1-Benzyl-3,4-bis(4-isopropoxyphenyl)-1H-pyrrole-2,5-dione
(26) 3,4-Dichloromaleimide 2 (400 mg, 1.6 mmol),
Table 3 Calculated (in vacuo) and experimental (in hexane) wavelengths of maximum absorption and emission of new compounds. Calculated
oscillator strengths are shown in parentheses
abs
max
abs
max
em
max
em
max
l
ðnmÞ calculated
l
ðnmÞ experimental
l
ðnmÞ calculated
l
ðnmÞ experimental
Compound
Dev. (%)
Dev. (%)
23
24
25
26
27
28
434.9 (0.530)
418.2 (0.496)
376.5 (0.450)
401.4 (0.476)
373.2 (0.579)
396.9 (0.478)
448a
474
344
440
363
406
−2.9
−11.8
9.4
552.3 (0.550)
568.2 (0.524)
477.3 (0.448)
521.2 (0.498)
473.5 (0.543)
517.5 (0.487)
575a
596
512
574
504
556
−3.9
−4.7
−6.8
−9.2
−6.1
−6.9
−8.8
2.8
−2.2
a in dichloromethane solvent

1084
J Fluoresc (2010) 20:1077–1085
~
(film): v ¼ 3; 011 cmꢀ1, 1,705 (C=O), 1,685 (C=O), 1,401,
1,266, 832, 752; MS (EI): m/z (%)=423.1 (100, [M]+),
408.1 (45), 380.1 (27), 91.0 (34). EI-HRMS calcd for
C27H21NO4 423.1471; found: 423.1465.
cysteine-modified diabodies targeting CD20 or HER2. Bioconjug
Chem 19:2527–2534
9. Hendricks RT, Sherman D, Strulovici B, Broka CA (1995) 2-aryl-
indolyl maleimides—novel and potent inhibitors of protein kinase
C. Bioorg Med Chem Lett 5:67–72
10. Kosower EM (1982) Intramolecular donor-acceptor systems. 9.
Photophysics of (phenylamino)naphthalenesulfonates: a paradigm
for excited-state intramolecular charge transfer. Acc Chem Res
15:259–266
11. Jones GII, Jackson WR, Choi C, Bergmark WR (1985) Solvent
effects on emission yield and lifetime for coumarin laser dyes.
Requirements for a rotatory decay mechanism. J Phys Chem
89:298–300
12. Van Gompel JA, Schuster GB (1989) Photophysical behavior of
ester-substituted aminocoumarins: a new twist. J Phys Chem
93:1292–1295
13. Takadate A, Masuda T, Murata C, Isobe A, Shinohara T, Irikura
M, Goya S (1997) A derivatizing reagent-kit using a single
coumarin fluorophore. Anal Sci 13:753–756
14. Matsunaga H, Santa T, Iida T, Fukushima T, Homma H, Imai K
(1997) Effect of the Substituent Group at the isothiocyanate
moiety of edman reagents on the racemization and fluorescence
intensity of amino acids derivatized with2, 1, 3-benzoxadiazolyl
isothiocyanates. Analyst 122:931–936
15. Saito R, Hirano T, Niwa H, Ohashi M (1997) Solvent and
substituent effects on the fluorescent properties of coelenteramide
analogues. J Chem Soc Perkin Trans 2:1711–1716
16. Fabian WMF, Niederreiter KS, Uray G, Stadlbauer W (1999)
Substituent effects on absorption and fluorescence spectra of
carbostyrils. J Mol Struct 477:209–220
17. Hutchison GR, Ratner MA, Marks TJ (2002) Accurate prediction
of band gaps in neutral heterocyclic conjugated polymers. J Phys
Chem A 106:10596–10605
1-Benzyl-3,4-bis(4-hydroxyphenyl)-1H-pyrrole-2,5-dione
(28) 3,4-Dichloromaleimide 2 (400 mg, 1.6 mmol) was
PdCl2(dppf).CH2Cl2 (191 mg, 0.23 mmol), 4-
hydroxyphenyl boronic acid (645 mg, 4.7 mmol), NaOH
(187 mg, 4.7 mmol, in 0.5 mL water) in THF (5 mL) was
degassed using the freeze pump thaw method. The ensuing
mixture was stirred at room temperature for 16 h. To the
ensuing mixture was treated with HCl (1.5 mL, 2 M) and
the aqueous layer was extracted with ether (4×20 mL). The
combined organic layers were dried (MgSO4) and concen-
trated under reduced pressure. The crude mixture was fused
to silica and passed through a plug of coarse silica with 1:1
ethyl acetate/hexane to remove the palladium catalyst. The
organic washings were treated with water (50 mL), K2CO3
(20 mL, sat aq). Treatment of the separated aqueous layer
(HCl, 20 mL, 2 M), extraction with ether (3×50 mL),
drying (MgSO4) and concentration of the organic layers
1
afforded diphenol 26 (110 mg, 18%) as an orange oil; H
NMR (300 MHz, d6-acetone): δ=7.46–7.20 (m, 9H, Ar–H),
6.90–6.82 (m, 4H, Ar–H), 4.80 (s, 2H, CH2; 13C NMR
(400 MHz, d6-acetone): δ=171.6, 159.6, 138.2, 134.9,
132.4, 129.3, 128.7, 128.2, 121.4, 116.2, 42.1; IR (KBr):
18. Wang BC, Liao HR, Yeh HC, Wu WC, Chen CT (2005)
Theoretical investigation of stokes shift of 3, 4-diaryl-substituted
maleimide fluorophores. J Lumin 113:321–328
19. Uchiyama S, Santa T, Imai K (1999) Semi-empirical PM3
calcualtion reveals the relationship between the fluorescence
characteristics of 4, 7-disubstituted benzofurazan compounds,
the LUMO energy and the dipole moment directed from the 4-
to the 7-position. Perkin Trans 2:569–576
v ¼ 3; 393 cmꢀ1, 1,693, 1,606, 1,351, 1,172, 838; MS (EI):
~
m/z (%)=371.1 (100, [M]+); EI-HRMS calcd for
C23H17NO4 371.1158; found: 371.0146.
20. Chachisvilis M, Chirvony VS, Shugla AM, Källebring B, Larsson
S, Sundström V (1996) Spectral and photophysical properties of
ethylene-bridged side-to-side porphyrin dimmers. 1. Ground-state
absorption and fluorescence study and calculation of electronic
structure of trans-1, 2-Bis(meso-octaethylporphyrinyl)ethane. J
Phys Chem 100:13857–13866
21. Amati M, Lelj F (2003) Luminescent Compounds fac- and mer-
Aluminum Tris(quinolin-8-olate). A pure and hybrid density
functional theory and time-dependent density functional theory
investigation of their electronic and spectroscopic properties. J
Phys Chem A 107:2560–2569
References
1. Landzettel WJ, Hargis KJ, Caboot JB, Adkins KL, Strein TG,
Veening H, Becker HD (1995) High-performance liquid chro-
matographic separation and detection of phenols using 2-(9-
anthrylethyl) chloroformate as a fluorophoric derivatizing reagent.
J Chromatogr A 718:45–51
2. Coates J (2001) Considerations in the fluorescence detection of
low levels of dissolved species in HPLC and other related liquid
flow techniques. Appl Spectrosc Rev 36:299–314
3. Wang YL (1989) Fluorescent analog cytochemistry: tracing
functional protein components in living cells. Methods Cell Biol
29:1–12
4. Small JV, Rottner K, Hahne P, Anderson KL (1999) Visualising
the actin cytoskeleton. Micros Res Tech 47:3–17
22. Chakraborty A, Kar S, Nath DN, Guchhait N (2006) Photoin-
duced intramolecular charge transfer reaction in (E)-3-(4-Methyl-
amino-phenyl)-acrylic acid methyl ester: a fluorescence study in
combination with TDDFT calculation. J Phys Chem A
110:12089–12095
5. Tsien RY, Miyawaki A (1998) Seeing the machinery of live cells.
Science 280:1954–1955
6. Selvin PR (2000) The renaissance of fluorescence resonance
energy transfer. Nat Struct Biol 7:730–734
23. Uray G, Kelterer AM, Hashim J, Glasnov TN, Kappe CO, Fabian
WMF (2009) Bisquinolones as chiral fluorophores—a combined
experimental and computational study of absorption and emission
characteristics. J Mol Struct 929:85–96, TDDFT
7. Kanaoka Y (1977) Organic fluorescence reagents in the study of
enzymes and proteins. Angew Chem Int Ed Engl 16:137–147
8. Sirk SJ, Olafsen T, Barat B, Bauer KB, Wu AM (2008) Site-
specific, thiol-mediated conjugation of fluorescent probes to
24. Sheikhshoaie I, Belaj F, Fabian WMF (2006) 1-(4-dimethylami-
nobenzyl)-2-(4-dimethylaminophenyl)-benzimidazole: Synthesis,
X-ray crystallography and density functional theory calculations.
J Mol Struct 794:244–250, TDDFT

J Fluoresc (2010) 20:1077–1085
1085
25. Cornelissen-Gude C, Rettig W (1999) An experimental and ab
initio CI study for charge transfer excited states and their relaxation
in pyrroloborane derivatives. J Phys Chem A 103:4371–4377
26. Yeh H-C, Wu W-C, Chen C-T (2003) The colourful fluorescence
from readily-synthesised 3, 4-diaryl-substituted maleimide fluo-
rophores. Chem Commun 3:404–405
27. Zerner MC, Leidlinger C, Fabian WMF, Junek H (2001) Push–
pull dyes containing malononitrile dimer as acceptor: synthesis,
spectroscopy and quantum chemical calculations. J Mol Struct
(Theochem) 543:129–146
28. Stewart SG, Polomska ME, Lim RW (2005) A concise synthesis
of maleic anhydride and maleimide natural products found in
Antrodia camphorata. Tetrahedron Lett 48:2241–2244
29. Dubernet M, Caubert V, Guillard J, Viaud-Massuard M-C (2005)
Synthesis of substituted bis(heteroaryl)maleimides. Tetrahedron
61:4585
30. Littke AF, Fu GC (2001) A versatile catalyst for Heck reactions of
aryl chlorides and aryl bromides under mild conditions. J Am
Chem Soc 123:6989–7000
35. Zerner M (1991) In: Lipkowitz KB, Boyd DB (eds) Reviews in
computational chemistry, vol 2. VCH, New York, p 313
36. Foresman JB, Head-Gordon MJ, Pople A, Frisch MJ (1992)
Toward a systematic molecular orbital theory for excited states. J
Phys Chem 96:135–149
37. Gaussian 03, Revision C.02, Frisch MJ, Trucks GW, Schlegel HB,
Scuseria GE, Robb MA, Cheeseman JR, Montgomery JA Jr,
Vreven T, Kudin KN, Burant JC, Millam JM, Iyengar SS, Tomasi
J, Barone V, Mennucci B, Cossi M, Scalmani G, Rega N,
Petersson GA, Nakatsuji H, Hada M, Ehara M, Toyota K, Fukuda
R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai
H, Klene M, Li X, Knox JE, Hratchian HP, Cross JB, Bakken V,
Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O,
Austin AJ, Cammi R, Pomelli C, Ochterski JW, Ayala PY,
Morokuma K, Voth GA, Salvador P, Dannenberg JJ, Zakrzewski
VG, Dapprich S, Daniels AD, Strain MC, Farkas O, Malick DK,
Rabuck AD, Raghavachari K, Foresman JB, Ortiz JV, Cui Q,
Baboul AG, Clifford S, Cioslowski J, Stefanov BB, Liu G,
Liashenko A, Piskorz P, Komaromi I, Martin RL, Fox DJ, Keith
T, Al-Laham MA, Peng CY, Nanayakkara A, Challacombe M,
Gill PMW, Johnson B, Chen W, Wong MW, Gonzalez C, Pople
JA (2004) Gaussian, Inc., Wallingford
31. El Yahyaoui A, Felix G, Heynderickx A, Moustrou C, Samat A
(2007) Convenient synthesis of photochromic symmetrical or
unsymmetrical bis(heteroaryl)maleimides via the Suzuki–Miyaura
cross-coupling reaction. Tetrahedron 63:9482–9487
32. Dewar MJS, Zoebisch EG, Healy EF (1985) AM1: a new general
purpose quantum mechanical molecular model. J Am Chem Soc
107:3902–3909
38. Ji, Jun SY, Qing Y, Liu S, Chen M, Zhao J (2009) Tuning the
intramolecular charge transfer of alkynylpyrenes: effecton photo-
physical properties and its application in design of OFF–ON
fluorescent thiol probes. J Org Chem 74:4855–4865
33. Zerner MC, Correa de Mello P, Hehenberger M (1982) Converging
SCF calculations on excited states. Int J Quantum Chem 21:251–259
34. Ridley JE, Zerner MC (1973) An intermediate neglect of
differential overlap technique for spectroscopy: pyrrole and the
azines. Theor Chem Acc 32:111–134
39. Ochi T, Yamaguchi Y, Wakamiya T, Matsubara Y, Yoshida Z-I
(2008) Block modification of rod-shaped π-conjugated carbon
frameworks with donor and acceptor groups toward highly
fluorescent molecules: synthesis and emission characteristics.
Org Biomol Chem 6:1222–1231