Synthesis of yellow and red fluorescent 1,3a,6a-triazapentalenes and the theoretical investigation of their optical properties

Article abstract of DOI:10.1039/c4sc02780a

To expand the originally developed fluorescent 1,3a,6a-triazapentalenes as fluorescent labelling reagents, the fluorescence wavelength of the 1,3a,6a-triazapentalenes was extended to the red color region. Based on the noteworthy correlation of the fluores

Full text of DOI:10.1039/c4sc02780a

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Synthesis of yellow and red fluorescent 1,3a,6a-
triazapentalenes and the theoretical investigation
of their optical properties†
Cite this: DOI: 10.1039/c4sc02780a
a
b
d
Kosuke Namba, Ayumi Osawa, Akira Nakayama, Akane Mera,b Fumi Tano,b
*
*
Yoshiro Chuman,c Eri Sakuda,c Tetsuya Taketsugu,c Kazuyasu Sakaguchi,c
c
c
*
Noboru Kitamura and Keiji Tanino
To expand the originally developed fluorescent 1,3a,6a-triazapentalenes as fluorescent labelling reagents,
the fluorescence wavelength of the 1,3a,6a-triazapentalenes was extended to the red color region. Based
on the noteworthy correlation of the fluorescence wavelength with the inductive effect of the 2-
substituent, electron-deficient 2-(2-cyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene and 2-
(2,6-dicyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene were synthesized. The former exhibited
yellow fluorescence and the latter exhibited red fluorescence, and both compounds exhibited large
Stokes shifts, and the 1,3a,6a-triazapentalene system enabled the same fluorescent chromophore to
cover the entire region of visible wavelengths. The potential applications of the 1,3a,6a-triazapentalenes
as fluorescent probes in the fields of the life sciences were investigated, and the 1,3a,6a-triazapentalene
system was clearly proven to be useful as a fluorescent reagent for live cell imaging. Quantum chemical
calculations were performed to investigate the optical properties of the 1,3a,6a-triazapentalenes. These
Received 10th September 2014
Accepted 9th October 2014
calculations revealed that the excitation involves
a significant charge-transfer from the 1,3a,6a-
triazapentalene skeleton to the 2-substituent. The calculated absorption and fluorescence wavelengths
showed a good correlation with the experimental ones, and thus the system could enable the theoretical
design of substituents with the desired optical properties.
DOI: 10.1039/c4sc02780a
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introducing uorophores is one of the most useful ways for
studying their mechanism.3 However, several key improvements
Introduction
are needed for the commonly used uorescent molecules. The
most highly uorescent molecules possess a relatively large
molecular size depending on the target bioactive compounds,
and the uorescence-labelled molecules sometimes lose their
activity as a result of the structural modications. Furthermore,
oen the methods used to synthesize them do not allow for the
design of systems whose luminescence properties span a wide
range of wavelengths. As a potential uorescent chromophore
to overcome the above problems, we have recently discovered
that a 1,3a,6a-triazapentalene skeleton without an additional
fused ring system is a compact and highly uorescent chro-
mophore.4,5 In contrast, benzotriazapentalene as an aryl-fused
ring system exhibits almost no uorescence (FF < 0.001),6 and
the various related analogues of the aryl-fused 1,3a,6a-tri-
azapentalenes7 have not been reported to have noteworthy
uorescence properties. The limited synthesis of 1,3a,6a-tri-
azapentalenes without an aryl-fused system8 might be the main
reason that they have been previously unrecognized as excellent
uorescent chromophores until our nding.
Fluorescent organic molecules are an important class of
compounds in modern science and technology, and are widely
used as biological imaging probes, sensors, lasers, and in light-
emitting devices.1 Thus, the development of useful uorescent
organic molecules is crucial for the advancement of many
industries, and has been a subject of intensive research.2 In
particular, small uorescent organic molecules have attracted
great attention in the eld of chemical biology, because the
visualization of biologically active small compounds by
aDepartment of Pharmaceutical Science, The University of Tokushima, 1-78 Shomachi,
Tokushima 770-8505, Japan. E-mail: namba@tokushima-u.ac.jp
bGraduate School of Chemical Sciences and Engineering, Hokkaido University,
Sapporo 060-0810, Japan
cDepartment of Chemistry, Faculty of Science, Hokkaido University, Kita-ku, Sapporo
060-0810, Japan
dCatalysis Research Center, Hokkaido University, Sapporo 001-0021, Japan
† Electronic supplementary information (ESI) available: the experimental details
for the synthesis of the triazapentalenes and the uorescent cell staining, the
absorption and uorescence spectra, and the 1H and 13C NMR spectra. Also
given are the molecular orbitals, the natural charges, the dipole moments, and
the Cartesian coordinates of the triazapentalenes (1a, 1b, 1g, 1e, and 1f). See
DOI: 10.1039/c4sc02780a
The construction of the 1,3a,6a-triazapentalene skeleton
without an aryl-fused ring system was recently established in
our laboratory, and the 1,3a,6a-triazapentalenes were readily
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prepared by the click-cyclization–aromatization cascade reac-
tion of various alkynes with the azide 2 possessing two triates
at the C2 and C3 positions (Scheme 1).4 The click reaction of
azide 2 with the alkynes produced triazole A, which underwent
cyclization to give triazolium ion B. In the presence of triethyl-
amine, the intermediate B was subsequently converted to tri-
azapentalene 1 by a sequential reaction of E2 elimination and
deprotonation (Scheme 1). This cascade reaction was conrmed
to be applicable to a wide range of alkynes, and the easy access
to the various 1,3a,6a-triazapentalenes was enabled. Further-
more, the 5,5-dimethoxy analog of B was found to be stable
enough for isolation, and a strong base was necessary for the
elimination of the methoxy group to give 5-methoxy-1,3a,6a-
triazapentalenes. This method was applicable to the one-pot
synthesis of the various 2,5-disubstituted-1,3a,6a-tri-
azapentalenes.9 Although the 1,3a,6a-triazapentalenes are
composed of a zwitter ion, the polarities and the electrical
charges are neutralised due to the resonance stabilization of the
aromatic compounds and so they are easily manipulated.
The 1,3a,6a-triazapentalenes exhibit not only intense uo-
rescence but also various interesting uorescence properties
such as an extremely large Stokes shi (Stokes shi exceeding
100 nm)10 and large positive uorescence solvatochromism.
More interestingly, the 1,3a,6a-triazapentalenes as uorescent
chromophores provide an innovative uorescence system that
can be tuned both in terms of the uorescence wavelength and
the quantum yield by varying the 2- and 5-substituents,
respectively.4,9 For example, the uorescence of the 1,3a,6a-tri-
azapentalenes shied to longer wavelengths due to the induc-
tive effect of the 2-substituents. In fact, the uorescence
maxima of the 2-phenyl-1,3a,6a-triazapentalene derivatives
exhibited a noteworthy correlation with the Hammett sp value
of the substituent on the benzene ring, as shown in Fig. 1. In
contrast, the introduction of an electron donating substituent at
the C5 position had little effect on the uorescence wavelength,
although the enhancement of the push–pull effect on the 10 p-
electron system was expected. Meanwhile, the uorescence
quantum yields (FF) were dramatically changed. In fact, the
introduction of a methoxy group at the C5 position of 2-(4-
Fig. 1 Substitution effect on the fluorescence properties of the
1,3a,6a-triazapentalenes. The values were determined in deaerated
dichloromethane.
cyanophenyl)-1,3a,6a-triazapentalene caused
a
substantial
increase in FF (from 0.15 to 0.57) without having any effect on
the uorescence wavelength .
Recently, emission- and/or quantum yield-tunable uo-
rophores have received a great deal of attention as the core
skeleton of uorescent probes.11 The 1,3a,6a-triazapentalene
system also provides a novel uorescent molecule that enables
the same uorescent chromophore to exhibit various uores-
cence colors and quantum yields. However, the detailed
mechanisms of the above interesting uorescence properties
have not been elucidated.
To actually develop the 1,3a,6a-triazapentalenes as uores-
cent labelling reagents, several goals had to be met: (i) to expand
the uorescence wavelength of the triazapentalenes to the red
color region, (ii) to conrm that the uorescence of tri-
azapentalene from the inside of cells is observable, (iii) to
introduce binding sites, such as a succinimide ester and a
maleimide moiety, as labelling reagents, and (iv) to obtain the
theoretical explanation of the uorescence properties of
1,3a,6a-triazapentalene. The uorescent labels exhibiting
longer emission wavelengths, such as those emitting yellow,
orange, and red light, might be more suitable for the living cells
and tissues due to the reduction of the light irradiation damage
and the potential access to deeper tissue. However, the existing
uorescent organic molecules emitting red light have several
common problems, including a large molecular size and a small
Stokes shi.11,12 On the other hand, 1,3a,6a-triazapentalene is a
compact uorescent chromophore exhibiting a large Stokes
shi, and its uorescence wavelength can be tuned based on
the inductive effect of C2-substituents. Although the uores-
cence wavelengths of the 1,3a,6a-triazapentalene derivatives
previously reported in a preliminary communication are below
the 556 nm (lime green) uorescence wavelength of 2-(4-nitro-
phenyl)-1,3a,6a-triazapentalene, additional introductions of
electron-withdrawing groups on the benzene ring are expected
to induce additional and longer wavelength shis. Thus, we
became intrigued by the synthesis of 1,3a,6a-triazapentalenes
Scheme 1 Single step synthesis of the 1,3a,6a-triazapentalenes (1).
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Table 1 The orientational effects of the cyano group on the benzene
ring in deaerated dichloromethane
possessing additional electron-withdrawing groups in order to
investigate the possibility of 1,3a,6a-triazapentalenes emitting
yellow, orange, and red light. Herein, we describe the synthesis
of 2-phenyl-1,3a,6a-triazapentalene derivatives possessing both
electron-withdrawing groups and binding sites on the benzene
ring, the observation of their uorescence inside cells, and the
computational efforts made to provide a theoretical explanation
of the uorescence properties of the 1,3a,6a-triazapentalenes
(Fig. 2).
max
abs
max
l
l
em
Yield (%) (nm) (nm) FF
Color
Results and discussion on the synthesis
and the fluorescence properties
77
87
381
327
509
493
0.15
0.24
A cyano group was chosen as the electron-withdrawing group
due to its small size and excellent stability under UV irradiation.
Thus, a suitable position for the introduction of the cyano group
to the benzene ring was rst investigated. Treatments of 2 with
the phenyl acetylene derivatives possessing a cyano group at the
para 3b, meta 3c, and ortho position 3d in the presence of the
CuI$ligand complex and triethylamine gave the desired tri-
azapentalenes 1b, 1c, and 1d with yields of 77%, 87%, and 93%,
respectively (Table 1).
93
376
515
0.24
In comparison with the para-substituent 1b, the introduc-
tion of a cyano group at the meta position (1c) induced the
undesired shorter wavelength shi, although the FF value was
increased to 0.24 (Table 1).13 In contrast, the ortho-cyano analog
1d exhibited a slightly longer-wavelength shi, and the FF value
was also increased (Table 1).13 Therefore, we found that the
ortho position is more suitable for the introduction of the cyano
group as an additional electron-withdrawing group for the
expansion of the uorescence wavelength to the yellow and red
color regions. Thus, we rst tried to synthesize methyl 3-cyano-
4-ethynylbenzoate 3e as an alkyne fragment. Commercially
available 5-bromo-2-iodobenzonitrile 4 was converted into
ethynylbenzonitrile 5 by the Sonogashira coupling reaction with
tert-butyldimethylsilylacetylene.14 The treatment of 5 with 5
mol% of Pd(PPh3)4 in methanol under a CO atmosphere
produced the methyl ester 6 in quantitative yield. Finally, the
removal of the TBS group gave the desired alkyne fragment 3e
(Scheme 2). Next, we tried to synthesize the dicyano analog,
which was expected to induce a further wavelength shi. 3,5-
Dicyano-4-iodo-benzoate 7 as a starting material was obtained
from the commercially available p-toluidine in 5 steps accord-
15
¨
ing to the procedure of Professor Gubel. The Sonogashira
coupling reaction of 7 with various acetylenes was initially
difficult, and yielded mainly the deiodinated reductive
product.16 Aer various investigations, we found that the reac-
tion with TBS–acetylene under the conditions of 10 mol% of
Pd2(dba)3$CHCl3, 20 mol% of trifurylphosphine, ꢀ20 mol% of
copper(I) iodide, and triethylamine in DMF at 50 C produced
the desired coupling product. Finally, the subsequent treatment
with TBAF and acetic acid gave the alkyne fragment 3f.
Fig.
2 Design of yellow and orange fluorescent 1,3a,6a-tri-
azapentalene and its application to cell staining.
Scheme 2 Synthesis of alkyne fragment 3e and 3f.
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Next, the cascade reaction leading to the production of the longer shi of the uorescence maximum in each case, and the
1,3a,6a-triazapentalenes was applied to the prepared alkynes 3e development of yellow and red uorescent 1,3a,6a-tri-
and 3f. The treatment of 3e with 1.2 equiv. of 2 in the presence azapentalenes was accomplished.
of 5 mol% of the CuI$ligand complex and triethylamine
It was especially noteworthy that these long-wavelength
produced the desired 1,3a,6a-triazapentalene 1e with a yield of uorescent molecules exhibited large Stokes shis, such as the
71%. The similar click reaction of 3f also proceeded smoothly to 152 nm shi of 1e and the 166 nm shi of 1f, despite there
give 1f with a yield of 72%. Furthermore, the comparative having been few prior examples of the long-wavelength ('550
analog 1g, which did not possess cyano groups, was also nm) organic uorophores exhibiting such large (mega) Stokes
synthesized with a yield of 73% from methyl 4-ethynylbenzoate shis,18 since such shis were useful for suppressing the action
(3g). Having prepared the desired 1,3a,6a-triazapentalenes 1e, of background uorescence in the various uorescence anal-
1f, and 1g, their uorescence properties were examined (Table yses. In addition, the molecular sizes of 1e and 1f were
2). Since these three compounds were only slightly soluble in considerably smaller in comparison with the conventional
water due to the lipophilicity of the benzene ring, their uo- yellow and red uorescent molecules. Therefore, the 1,3a,6a-
rescence spectra were measured in deaerated dichloromethane. triazapentalenes might be practical uorescent chromophores
The standard analog 1g exhibited a high uorescence quantum for use as molecular probes to cover the entire region of visible
max
yield (FF ¼ 0.44)13 and green emission (l
¼ 510 mm) as wavelengths, although the further shi toward longer wave-
em
predicted from the Hammett sp value of the methyl ester on the lengths is still needed. Furthermore, 1g and 1e exhibited uo-
benzene ring. As we expected, the mono-cyano analog 1e rescence emission in the solid state with a uorescence
showed a noteworthy longer-wavelength shi of the uores- maximum similar to that observed in the solution of dichloro-
cence maximum from 510 nm of 1g to 572 nm, and 1e emitted methane, whereas the uorescence of 1f in the solid state was
yellow light. Although the uorescence quantum yield (FF) of 1e not detected.
was slightly decreased to 0.34,17 this value was still within the
On the other hand, the extinction coefficient (3) of 1g at 376
range required for an effective uorescent labelling reagent. nm was 1230 dm3 molꢁ1 cmꢁ1, and this value still needed to be
Furthermore, the uorescence maximum of the di-cyano analog increased for a more bright uorescent reagent. Although the 3
1f shied to a still longer-wavelength region (632 nm), and 1f value at 287 nm was 13 800 dm3 molꢁ1 cmꢁ1, at a practical level,
exhibited red uorescence. Therefore, the introductions of the this region (ultraviolet) is not a suitable excitation light for
cyano groups were found to induce an approximately 60 nm imaging probes. Similarly, the extinction coefficients (3) of 1e
Table 2 Yields and fluorescence properties of 1g, 1e, and 1f
1g
1e
1f
73%
71%
72%
Yield
Solutiona
Solid
Solutiona
Solid
Solutiona
lmabasx (nm)
lmemax (nm)
FF
376
510
0.44
N/A
496
0.06
420
572
0.34
N/A
549
0.06
466
632
0.096
Color
a
In deaerated dichloromethane.
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and 1f in a visible light region were also not high at 630 dm3
molꢁ1 cmꢁ1 (420 nm) and 1580 dm3 molꢁ1 cmꢁ1 (466 nm),
respectively. Therefore, the improvement of the extinction
coefficient (3) was the next challenge for the development of
more useful bright uorescent labels. So far, we have already
found that the introduction of a substituent at the C4 position
dramatically increases the 3 value. For example, 4-phenyl
analogs of 1g showed a substantial increase in the 3 value from
1230 dm3 molꢁ1 cmꢁ1 (376 nm) for 1g to 22 600 dm3 molꢁ1
cmꢁ1 (345 nm) with comparable FF values. The 4-phenyl analog
of 1e also exhibited a practical 3 value of 4560 dm3 molꢁ1 cmꢁ1
(432 nm) and 38 000 dm3 molꢁ1 cmꢁ1 (336 nm), although the
FF value was decreased to 0.07.19 Further investigation of 4-
substituents for the design of practical uorescent labelling
reagents is currently underway in our laboratory.
Furthermore, the uorescence solvatochromism of 1e was
examined. The uorescence spectra of 1e in several solvents are
shown in Fig. 3. Basically, the uorescence of 1e shied to the
longer wavelength with the Stokes shi being increased by an
increase in the solvent polarity from benzene (546 nm) to
Fig. 4 The observation of 1e in HeLa cells. Living cells were cultured in
0.02% DMSO as a control (a and b) or with 10 mM 1e in 0.02% DMSO (c
and d). The uptake of 1e was monitored by using a fluorescence
microscope (BZ-9000; Keyence) with a bright-field image (b and d) or
with a fluorescence image (a and c) with a BZ set (FF01-452/45 nm
exciter, FF01-607/70 nm emitter, FF511 nn-Di01 dichroic mirror).
acetone (645 nm). On the other hand, its uorescence in
max
em
methanol shied inversely to the shorter wavelength (l
¼
463 nm). Furthermore, since 1e was only slightly soluble in
water, its uorescence in water was also measured. The uo-
rescence shied to 476 nm similarly to the uorescence shi
observed in methanol. The uorescence quantum yield (FF) in
water was substantially decreased to a value of 0.013. Therefore,
the 1,3a,6a-triazapentalenes are expected to change their uo-
rescence wavelength and intensity according to the hydro-
phobic environment in the cells.
uorescent 1e by living cells and the uorescence sol-
vatochromism of 1e enhance the uorescence contrast between
the cells and the background, it was not necessary to x the
cells. Furthermore, a cytotoxic effect on the cells was not iden-
tied over the observation period, suggesting that the tri-
azapentalene is suitable for connecting to small biofunctional
molecules as a uorescent label. This is the rst experimental
evidence that the 1,3a,6a-triazapentalene is applicable to the life
sciences eld as a uorescent reagent. Further detailed inves-
tigations into the localization and quantitative analysis of 1e
inside cells are currently underway in our laboratory.
The actual uorescence observation of 1e inside cells
encouraged us to develop 1e as a uorescent labelling reagent.
Thus, the conversion of the methyl ester moiety into the N-
hydroxysuccinimide ester as a binding site was attempted. The
treatment of 1e with 1.2 equiv. of lithium hydroxide afforded
carboxylic acid 8, which was directly used for the next conden-
sation reaction. However, although the condensation reaction
proceeded smoothly, the removal of the urea analogs generated
from the condensing reagent was not straightforward due to the
instability of the succinimide moiety of 9. Finally, polymer-
supported DCC was adopted as a useful condensing reagent to
remove the urea by ltration, and the subsequent recrystalli-
zation gave the puried 9 in a 60% two-step yield. Having
prepared the uorescent labelling reagent 9, the introduction of
9 into amino acids was examined (Scheme 3). The treatment of 9
with glycine ethyl ester in DMF produced labelled glycine 10 in
Next, we investigated the applicability of the long-wavelength
uorescent 1,3a,6a-triazapentalenes as uorescent probes in a
biological system. Since the di-cyano analog 1f was not very
stable under UV irradiation and its FF was lower (FF ¼ 0.096)
than that of 1e (FF ¼ 0.34),17 the mono-cyano analog 1e was
adopted for this purpose. Thus, HeLa cells were treated with a
solution of 1e (10 mM in 0.02% DMSO)20 and monitored in the
572–642 nm wavelength region. As shown in Fig. 4, the uo-
rescent staining of HeLa cells was successfully observed without
washing the cellular medium. The living HeLa cells were clearly
visualized as observed using
a uorescence microscope,
whereas the interiors of the control cells, which were treated
with DMSO, were not stained. Since the active uptake of the
95% yield. The uorescence-labelled 10 exhibited yellow emis-
max
em
sion (l
¼ 567 nm) with a high quantum yield (FF ¼ 0.37)17 in
Fig. 3 Emission behaviors and the fluorescence spectra of 1e in
several solvents.
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dependent density functional theory (TD-DFT), but in this work
the high-level wavefunction-based approach using the complete
active space second-order perturbation theory (CASPT2) method
are also employed to provide a more reliable description of the
excitation energies. The following synthetic 1,3a,6a-tri-
azapentalenes were examined as the model substrates in this
investigation: unsubstituted 1,3a,6a-triazapentalene 1a as a
basic structure, 2-(4-cyano)phenyl derivative 1b as a standard
analog described in the previous communication, and synthetic
1g, 1e, and 1f as described in this article.
Computational details
The equilibrium geometry in the electronic ground state (S0) is
determined by the density functional theory (DFT) calculations
using the B3LYP functionals, while the geometry optimization
in the lowest pp* excited state S1(pp*) is performed by the
time-dependent DFT (TD-DFT) calculations employing the
coulomb attenuated B3LYP (CAM-B3LYP) functionals.22 The Cs
symmetry constraint is imposed for 1a, 1b, 1g, and 1e, while no
constraint is applied for 1f because the twisted structure is more
stable due to the steric hindrance. The choice to employ the
CAM-B3LYP functionals is due to the signicant charge-transfer
character involved in excitation to the S1 state. The 6-31 + G(d,p)
basis set is used in the DFT calculations and the equilibrium
geometries are determined both in the gas phase and in
dichloromethane. The solvent effects are taken into account by
the polarizable continuum solvation model (PCM),23 where the
radii are taken from the universal force eld.24 Aer the
geometry optimization, the vertical excitation and uorescence
energies are calculated at the S0 and S1 equilibrium structures
(denoted as (S0)min and (S1)min), respectively, by the TD-
DFT(CAM-B3LYP) method. In PCM calculations, the linear-
response method with a non-equilibrium solvation is employed
to obtain the vertical excitation energies at (S0)min, while the
equilibrium solvation is adapted for the calculation of the
excitation energies during the S1 geometry optimization.
The excitation energy is also rened at the DFT-optimized
geometries by the CASPT2 (ref. 25) method in order to obtain
more reliable excitation energies. A level shi with a value of 0.3
is applied for the CASPT2 calculations.26 The notation of
CASPT2 (m,n) is occasionally used, in which case the active
space for a reference state-averaged complete active space self-
consistent eld (SA-CASSCF) wavefunction is composed of m
electrons and n orbitals (SA-CASSCF (m,n)). The augmented
correlation-consistent polarized double-zeta basis set (denoted
as aug-cc-pVDZ) is employed in the CASPT2 calculations. For
obtaining the oscillator strengths, the vertical excitation ener-
gies calculated by CASPT2 and the transition dipole moments
Scheme 3 The synthesis of the fluorescent labelling reagent 11 and its
application to the amino acids. Measured in water.
a
deaerated dichloromethane. Furthermore, the introduction of
the tri-peptide Gly-Pro-Leu was also examined, and the labelled
tri-peptide 11 was obtained in 82% yield. The uorescence
observation of 11 showed a uorescence maximum at 567 nm
and an acceptable uorescence quantum yield (FF ¼ 0.24)17 in
deaerated dichloromethane. Therefore, the development of the
1,3a,6a-triazapentalene as a compact uorescent labelling
reagent emitting yellow-red light was achieved. Furthermore,
although the labelled glycine 10 and tri-peptide 11 were dis-
solved well in an organic solvent,21 their uorescence properties
in water were also measured. Since the emission maxima of 10
and 11 shied to shorter wavelengths with similar absorption
maxima, the Stokes shis became small in water as in the case
of 1e. The uorescence quantum yields (FF) were also reduced
to 0.019 (10) and 0.077 (11).17 These changes in the uorescence
properties according to the polarity of the environment might
make the 1,3a,6a-triazapentalene useful as a uorescent probe
in vivo measurements.
Theoretical investigation of the optical properties of the
1,3a,6a-triazapentalenes
In our preliminary communication, we rst reported that the calculated by SA-CASSCF are used.
1,3a,6a-triazapentalene skeleton without an additional fused
For 1a, the active space for the reference SA-CASSCF wave-
ring system is a compact and highly uorescent chromophore. function is comprised of six p orbitals (four p orbitals are
However, the detailed mechanisms of the uorescence have not doubly-occupied and two are unoccupied in the closed-shell
yet been elucidated. In this work, quantum chemical calcula- conguration), and it is therefore denoted as SA-CASSCF(8,6).
tions were performed to investigate the optical properties of the 1a possesses ten p orbitals and the lowest and highest p
1,3a,6a-triazapentalenes. Most of the theoretical calculations orbitals are excluded from the active space. This is justied by
for the optical properties of dye molecules utilize the time- the larger active space calculation, which includes all p orbitals
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(which corresponds to SA-CASSCF(10,8), and the active orbitals 4.31 eV (288 nm), even though the experimental measurements
at (S0)min are shown in the ESI as Fig. S1†), where only a are performed in dichloromethane. The second pp* excited
difference of ꢂ0.01 eV is observed in the S1 vertical excitation state is characterized by the HOMO / LUMO+1 transition, and
energies. The active space for the other chromophores is it lies close to the rst pp* state in the CASPT2 calculation. The
composed of twelve electrons distributed in ten p orbitals (SA- natural charges of the S0 and S1 states at (S0)min and their
CASSCF(12,10)), and the active orbitals of 1b at (S0)min are differences are shown in Fig. S3.†
shown in Fig. S2.† As seen in the gure, the active space of the
SA-CASSCF(12,10) wavefunction includes orbitals that corre-
spond to the active orbitals of SA-CASSCF(8,6) in 1a. For all
chromophores, the S0 and S1 states are averaged with equal
weights in the SA-CASSCF calculations, except where otherwise
noted.
The DFT and TD-DFT calculations are performed using the
Gaussian09 program package27 while the CASPT2 calculations
are carried out using the MOLPRO2010.1 program package.28
2. 2-(4-Cyano)phenyl-1,3a,6a-triazapentalene (1b)
The S0 and S1 equilibrium structures of 1b in the gas phase are
shown in Fig. 6, along with the bond lengths and atomic
numbering. The transition to the S1 state involves the bond
˚
elongation of C7–C8 (1.394 / 1.444 A) and shortening of the
˚
central C8–C13 bond (1.469 / 1.421 A).
The vertical excitation energies to the low-energy-lying pp*
states are shown in Table 4. In the CASPT2 calculation, the S0
and the lowest three pp* states are averaged with equal weights
in the reference SA-CASSCF(12,10) wavefunction. The vertical
excitation energies to the S1(pp*) state are 3.85 (322 nm) and
3.25 eV (381 nm) for the TD-DFT and CASPT2 calculations,
respectively, and the CASPT2 excitation energy is in remarkably
good agreement with the experimental value of 3.25 eV (381 nm)
(note again that the experimental measurements are performed
in dichloromethane). Excitation to the S1 state is characterized
by the HOMO / LUMO transition (see Fig. 7 and also Fig. S2†),
and as expected from the shape of the two relevant orbitals, the
S1 transition involves charge transfer from the 1,3a,6a-tri-
azapentalene skeleton to the substituted phenyl ring. This is
clearly seen from the large dipole moment in the S1 state (19.47
debye) compared to that of the S0 state (7.11 debye) at (S0)min
(see Table S1†). The charge-transfer character of S1 is also clear
from the natural charges, where the sums of the natural charges
in the 1,3a,6a-triazapentalene skeleton (atoms from N1 to H12)
are 0.022 and 0.542 in the S0 and S1 states, respectively (see also
Table S1†). Since the S1 state exhibits a charge-transfer char-
acter, it may be possible to observe the twisted intramolecular
charge transfer (TICT) state involving the rotation of the phenyl
ring around the central C8–C13 bond. In order to check this, we
performed frequency analysis at (S1)min and conrmed that the
planar geometry is the minimum energy structure in the S1
state.
Results and discussion on the optical
properties
We begin by investigating the character of the excited states of
1a and 1b at (S0)min in the gas phase, followed by the results and
discussion on the optical properties of the other chromophores
in the gas phase and in dichloromethane.
1. Simple 1,3a,6a-triazapentalene (1a)
The S0 and S1 equilibrium structures of 1a in the gas phase are
shown in Fig. 5, along with the bond lengths and the atomic
numbering (note that this numbering is different from the
previous sections and is only used in the theoretical section).
The signicant changes in geometry upon photo-excitation
˚
involve the bond elongation of N3–C6 (1.370 / 1.411 A) and
˚
N1–N2 (1.344 / 1.376 A).
The vertical excitation energies to the low-energy-lying pp*
states are shown in Table 3, where in the CASPT2 calculation the
S0 and lowest three pp* states are averaged with equal weights
in the reference SA-CASSCF(8,6) wavefunction. It is noted that,
although a couple of np* states are found between these pp*
states in the TD-DFT calculations, it is conrmed that the
lowest-energy singlet excited-state is characterized by the pp*
excitation, and therefore only the pp* states are examined in
this investigation. The lowest pp* excited-state, S1(pp*), is
viewed as the HOMO–LUMO transition (see the natural orbitals
in Fig. S1 (ESI)†) and the CASPT2 excitation energy of 4.33 eV
(286 nm) is in good agreement with the experimental value of
As seen in Table 4, the second and third pp* states can be
described as a mixing of two congurations, HOMO /
LUMO+1 (9p / 2p*) and HOMOꢁ1 / LUMO (8p / 1p*). It is
noted that the HOMO / LUMO+1 (9p / 2p*) transition
corresponds to the S0–S1 excitation of 1a, while the S0–S1 tran-
sition of 1b corresponds to the excitation to the second pp*
state of 1a (see the natural orbitals given in Fig. S1 and S2†).
Therefore, the electronic character of the S1 state is different
between 1a and 1b.
3. Green uorescence (1g), yellow uorescence (1e), and red
uorescence (1f) derivatives and comparison with the
experimental results
The optimized structures of 1f in the S0 and S1 states are shown
in Fig. 8, where the dihedral angle of d(C7–C8–C13–C15) rep-
resenting twisting of the phenyl ring around the central C8–C13
Fig. 5 The equilibrium structures of 1a in the S0 and S1 states in the gas
phase. The bond lengths are given in units of A˚.
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a
Table 3 The vertical excitation energies (DE in eV and nm) and oscillator strengths (f in a.u.) of 1a for the low-lying pp* states at (S0)min
TD-DFT (CAM-B3LYP)
CASPT2
State
DE (eV)
DE (nm)
f
Transition
DE (eV)
DE (nm)
f
Transition
1
2
3
4.79
5.33
5.49
259
232
225
0.262
0.052
0.010
5p / 1p*
5p / 2p*
5p / 3p*
4.33
4.43
5.53
286
280
224
0.213
0.250
0.384
5p / 1p*
5p / 2p*
4p / 1p*
a
The main orbital transition is also shown.
Fig. 7 Natural orbitals of 1b involved in the excitation to the S1 state.
Fig. 6 Equilibrium structures of 1b in the S0 and S1 states in the gas
phase. The bond lengths are given in units of A˚.
atoms is 40.6 degrees at (S0)min, and slightly decreases to 29.7
degrees at (S1)min. The other chromophores (1g and 1e) main-
tain the planar geometry, and the Cartesian coordinates of the
optimized structures are given in the ESI.†
Excitation to the S1 state involves the HOMO / LUMO
transition, and all chromophores (1g, 1e, and 1f) exhibit the
same charge-transfer character. The vertical excitation and
uorescence energies are summarized in Table 5 and Table 6,
respectively. We note here that in this table a slight discrepancy
is found in the S1(pp*) vertical excitation energies of CASPT2
for 1a and 1b with respect to the values shown in Table 3 and 4,
since in Table 5 only the S0 and S1 states are averaged with equal
weights in the reference SA-CASSCF wavefunction.
Fig. 8 The equilibrium structures of 1f in the S0 and S1 states in the gas
phase. The bond lengths are given in units of A˚.
dichloromethane using the solvatochromic shis of TD-DFT
calculations (the estimated values are shown in parenthesis).
Fig. 9 shows the comparison of absorption and uorescence
wavelengths between the theoretical calculations and experi-
mental results. Although the calculated uorescence wave-
lengths are shorter than the experimental values, the gure
The CASPT2 calculations are performed only in the gas
phase, and therefore we estimate the excitation energies in
a
Table 4 The vertical excitation energies (DE in eV and nm) and oscillator strengths (f in a.u.) of 1b for the low-energy-lying pp* states at (S0)min
TD-DFT (CAM-B3LYP)
CASPT2
State
DE (eV)
DE (nm)
f
Transition
DE (eV)
DE (nm)
f
Transition
1
2
3
3.85
4.67
4.80
322
266
258
0.083
0.043
1.111
9p / 1p*
9p / 2p*, 8p / 1p*
9p / 2p*, 8p / 1p*
3.25
3.89
4.17
381
319
298
0.047
0.755
0.012
9p / 1p*
9p / 2p*, 8p / 1p*
9p / 2p*, 8p / 1p*
a
The main orbital transition is also shown.
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Table 5 The vertical excitation energies (DE in eV and nm) and oscillator strengths (f in a.u.) for the S1 state calculated by TD-DFT and CASPT2 at
(S0)min
TD-DFT (in gas)
TD-DFT (in dichloromethane)
CASPT2 (in gas)
Exp.
DE (eV)
DE (nm)
f
DE (eV)
DE (nm)
f
DE (eV)
DE (nm)
f
DE (nm)
1a
1b
1g
1e
1f
4.79
3.85
3.91
3.51
3.21
259
322
317
353
387
0.262
0.083
0.096
0.048
0.033
4.62
3.66
3.73
3.33
3.11
268
339
332
372
398
0.276
0.143
0.159
0.076
0.050
4.26
3.26
3.32
2.97
2.76
291 (300)a
380 (397)
374 (389)
418 (437)
450 (461)
0.351
0.058
0.057
0.051
0.040
288
381
376
420
466
a
The number in parenthesis is an estimate in dichloromethane.
clearly demonstrates a good correlation between the two values.
The overestimation of the uorescence energies may be attrib-
uted to the insufficient treatment of the solvent environments,
because excitation involves a signicant charge-transfer char-
acter. The explicit treatment of the solvent molecules in the
framework of the QM/MM approach or the state-specic
approach29,30 would be appropriate for a more quantitative
description of the uorescence energies.
In the ESI (Tables S1 and S2†), the sums of the natural
charges in the 1,3a,6a-triazapentalene skeleton and the dipole
moments in the S0 and S1 states at (S0)min and (S1)min are given
for all chromophores. It is noteworthy that there is a clear
correlation between the wavelengths and the natural charges
(also the dipole moments) in the S1 state, where a larger charge
separation induces longer absorption and uorescence wave-
lengths. It is also noted that the absorption and uorescence
wavelengths are longer when measured in dichloromethane
than when measured in the gas phase because the charge-
transfer state is more stabilized in polar solvents.
Fig. 9 The comparison of (a) the absorption and (b) the fluorescence
wavelengths between the theoretical calculations and experimental
results. The central line indicates a perfect theory/experiment match.
Conclusions
The uorescence wavelengths of 1,3a,6a-triazapentalenes were
extended to the red color region. Based on the noteworthy
Finally, we comment that the CASPT2 method is more reli- correlation of the uorescence wavelength with the inductive
able than the TD-DFT approach, but the computational cost is effect of the 2-substituent, electron decient 2-(2-cyano-
much more expensive. As seen in the present work, the TD-DFT
method predicts slightly higher excitation energies than those
by CASPT2, but the correlation with experimental results is
surprisingly good. Therefore, for chromophores of a larger size,
where the computational costs of CASPT2 calculations are
4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene and 2-(2,
6-dicyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene
were synthesized. They exhibited yellow and red uorescence
and a large Stokes shi respectively, and the 1,3a,6a-tri-
azapentalene system enabled the same uorescent chromo-
prohibitive, the TD-DFT method can be reliably used to predict phore to cover the entire region of visible wavelengths. The
the optical properties of 1,3a,6a-triazapentalenes.
potential applications of the 1,3a,6a-triazapentalene system as
uorescent probes in the elds of the life sciences were
Table 6 The vertical fluorescence energies (DE in eV and nm) and oscillator strengths (f in a.u.) for the S1 state calculated by TD-DFT and CASPT2
at (S1)min
TD-DFT (in gas)
TD-DFT (in dichloromethane)
CASPT2 (in gas)
Exp.
DE (eV)
DE (nm)
f
DE (eV)
DE (nm)
f
DE (eV)
DE (nm)
f
DE (nm)
1a
1b
1g
1e
1f
4.62
3.46
3.48
3.14
2.82
268
358
356
394
440
0.276
0.137
0.164
0.072
0.046
4.33
3.18
3.19
2.91
2.68
287
390
389
425
463
0.446
0.345
0.372
0.191
0.134
4.04
2.88
2.91
2.63
2.33
307 (326)a
430 (462)
426 (459)
471 (502)
533 (556)
0.382
0.092
0.101
0.078
0.060
389
509
510
572
632
a
The number in parenthesis is an estimate in dichloromethane.
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investigated, and the 1,3a,6a-triazapentalene system was clearly
proven to be useful as a uorescent reagent for living cells. The
N-hydroxysuccinimide ester derivative of yellow uorescent
1,3a,6a-triazapentalene as a compact labelling reagent was
conrmed to be able to readily label the amino group. Finally,
quantum chemical calculations were performed to investigate
the optical properties of the 1,3a,6a-triazapentalenes. These
calculations revealed that excitation involves signicant charge-
transfer from the 1,3a,6a-triazapentalene skeleton to the 2-
substitutent. The calculated absorption and uorescence
wavelengths showed a good correlation with the experimental
ones, which allows us to design substituents that exhibit the
desired optical properties.
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Acknowledgements
We thank Professors Kazuki Sada and Kenta Kokado for the
TGA analysis of 2. This work was partially supported by Grant-
in-Aid for Scientic Research (Grant no. 24310162), and Grant-
in-Aid for Scientic Research on Innovative Areas (Project no.
2301: Chemical Biology of Natural Products) from the Ministry
of Education, Culture, Sports, Science, and Technology, Japan.
K. N. is grateful to the Naito Foundation and the Yamada
Foundation for support through a Research Fund for Recently
Independent Professor. A. O. is grateful to JSPS for a Research
Fellowship (no. 26 2457) for Young Scientists.
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W. E. Piers, B. Heyne and R. McDonald, Angew. Chem., Int. 20 The uorescence properties of 1e in this solution (0.02%
max
max
Ed., 2011, 50, 12214–12217, and references are therein.
DMSO); l
¼ 340 nm, l
¼ 485 nm, FF ¼ 0.035.
abs
em
11 For emission tunable uorophores, see (a) A. P. Demchenko, 21 The lithium salt of 11 obtained by hydrolysis of methyl ester
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and S. B. Park, Angew. Chem., Int. Ed., 2014, 53, 1346–1350;
moiety (C-terminus) was readily dissolved in water. The
max
uorescent properties of lithium salt in water; l
¼ 390
abs
max
em
nm, l
¼ 524 nm, FF ¼ 0.0016.
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13 Fluorescence quantum yields were estimated by using 9,10-
diphenylanthracene (9,10-DPA) in cyclohexane (FF ¼ 0.91) as
a standard.
14 Although the use of trimethylsilylacetylene also gave the
coupling product, the TMS group was cleaved under the
condition of next CO insertion reaction.
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click reaction of 1-phenyl-substituted azidoditriate 12 with
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