Study of Model Systems for Bilirubin and Bilin Chromophores: Determination and Modification of Thermal and Photochemical Properties

Article abstract of DOI:10.1021/acs.joc.6b00892

Bilin chromophores and bilirubin are involved in relevant biological functions such as light perception in plants and as protective agents against Alzheimer and other diseases. Despite their extensive use, a deep rationalization of the main factors controlling the thermal and photochemical properties has not been performed yet, which in turn hampers further applications of these versatile molecules. In an effort to understand those factors and allow control of the relevant properties, a combined experimental and computational study has been carried out for diverse model systems to understand the interconversion between Z and E isomers. In this study, we have demonstrated the crucial role of steric hindrance and hydrogen-bond interactions in thermal stability and the ability to control them by designing novel compounds. We also determined several photochemical properties and studied the photodynamics of two model systems in more detail, observing a fast relaxation of the excited state shorter than 2 ps in both cases. Finally, the computational study allowed us to rationalize the experimental evidence.

Full text of DOI:10.1021/acs.joc.6b00892

Article
pubs.acs.org/joc
Study of Model Systems for Bilirubin and Bilin Chromophores:
Determination and Modification of Thermal and Photochemical
Properties
Cristina García-Iriepa,‡ Hanna A. Ernst,§ Yu Liang,§ Andreas-Neil Unterreiner,§ Luis Manuel Frutos,‡
and Diego Sampedro*,†
†Departamento de Química, Centro de Investigacion
E-26006 Logrono, Spain
́
en Síntesis Química (CISQ), Universidad de La Rioja, Madre de Dios 53,
̃
‡Unidad Docente de Química Física, Universidad de Alcala,
́
Alcala de Henares, E-28871 Madrid, Spain
́
§Institute of Physical Chemistry, Karlsruhe Institute of Technology (KIT), 76128 Karlsruhe, Germany
S
* Supporting Information
ABSTRACT: Bilin chromophores and bilirubin are involved in
relevant biological functions such as light perception in plants and as
protective agents against Alzheimer and other diseases. Despite their
extensive use, a deep rationalization of the main factors controlling
the thermal and photochemical properties has not been performed
yet, which in turn hampers further applications of these versatile
molecules. In an effort to understand those factors and allow control
of the relevant properties, a combined experimental and computa-
tional study has been carried out for diverse model systems to
understand the interconversion between Z and E isomers. In this
study, we have demonstrated the crucial role of steric hindrance and
hydrogen-bond interactions in thermal stability and the ability to
control them by designing novel compounds. We also determined
several photochemical properties and studied the photodynamics of
two model systems in more detail, observing a fast relaxation of the excited state shorter than 2 ps in both cases. Finally, the
computational study allowed us to rationalize the experimental evidence.
methine units, leading to a structure with extended conjugation
(see Figure 1A).15
INTRODUCTION
■
Photoisomerization of carbon−carbon double bonds is a
common mechanism in biological processes. The different
biological activities and properties of Z and E isomers lead to
diverse functions and responses.1−5 Taking advantage of this,
the use of light has been extensively employed in different
therapies, so-called phototherapies, as a way to control the Z/E
isomer ratios of certain chromophores with quite different
biological cytotoxicities.6−9 Well-known chromophores that
involve Z/E photoisomerizations in nature are the retinal
chromophore in rhodopsins, which controls the process of
vision,2,10 and the green fluorescent protein (GFP) chromo-
phore,4,11,12 extensively applied as a reporter gene and
fluorescent probe.
Another large and heterogeneous family of Z/E photo-
switchable biomolecules is phytochromes, photoreceptors
present not only in plants but also in some bacteria and
fungi.13,14 These proteins consist of two major structural
domains, the globular N-terminal chromophore-binding
domain (65 kDa) and the structurally open C-terminal domain.
The photoactive part includes a bilin chromophore with an
open tetrapyrrolic system in which the rings are connected by
This chromophore can be interconverted between two
different states, the dark-stable, red-absorbing isomer Pr and the
metastable far-red-absorbing isomer Pfr. Upon exposure to light
(λmax = 660 nm), Pr undergoes photoisomerization of one
exocyclic carbon−carbon double bond followed by proton
transfer, leading to Pfr (λmax = 730 nm), which is usually the
physiological active isomer.16 The biological output, Pr/Pfr ratio,
depends not only on the ambient light but also on the
photochemical reaction kinetics and the thermal (dark)
reversion process.17,18 This photoisomerization leads to a
reversible conformational change of the protein,19 which
constitutes the first step of the phytochrome-signaling cascade.
This basic system allows for the regulation of several functions
such as seed germination or photomorphogenesis in plants20 or
growth, development, and pigmentation in bacteria.21−24
In humans, bilin chromophores are metabolic products of
some porphyrins, such as bilirubin, whose chemical structure is
very similar to those of bilin pigments (see Figure 1B).25−27
Received: April 20, 2016
© XXXX American Chemical Society
A
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strength of hydrogen-bond interactions, rationalized the effect
of this stabilization energy on the relative thermal stability of
both isomers, and demonstrated their effect on the photo-
chemical properties (e.g., UV−vis spectra, photoisomerization
extent). Furthermore, in order to better understand the
biological processes, we aimed to figure out the photo-
isomerization dynamics and mechanism of two representative
models. The dynamic processes that take place during the
photoisomerization were studied by UV−vis transient
absorption spectroscopy (TAS). In addition, a computational
study was performed to investigate the geometry of the ground-
state minima, the photoisomerization mechanism and the
hydrogen bond effect in the excited state. The data collected
allowed us to rationalize the properties of these model systems
and provide valuable information for the more complex
biological systems.
RESULTS
■
Synthesis and Thermal Properties. First, benzalpyrroli-
none derivatives (compounds 1a−j) were synthesized following
the method of Chepelev et al.39 (see Scheme 1A). The selected
Figure 1. Structures of (A) phytochrome and (B) bilirubin
chromophores. The photoisomerizable double bonds and their
corresponding model systems are depicted.
The study of the photophysical and photochemical properties
of bilirubin is of special interest as its elevated levels are related
to diverse diseases such as neonatal jaundice among others.
Phototherapy is the treatment most used against this
affliction.28−30 Similarly to bilin chromophores, an exocyclic
carbon−carbon double bond of the dark stable ZZ isomer
photoisomerizes to the metastable ZE isomer after irradiation.31
This photoproduct presents a more polar chemical structure, as
some of the hydrogen bonds present in the ZZ isomer are
disrupted, facilitating its excretion across the liver to the
bile.31,32
Scheme 1
The study of model systems of photoswitchable biological
chromophores is an approach extensively applied to understand
the properties of the natural analogues. For instance, the penta-
2,4-dieniminium cation and 4-(4-hydroxybenzylidene)-1,2-
dimethyl-1H-imidazol-5(4H)-one have been used in numerous
studies as model systems of the retinal33,34 and the GFP
chromophores,35−38 respectively. Concerning the phytochrome
chromophore and bilirubin, dipyrrinone39,40 and benzalpyrro-
linone41,42 derivatives (see Figure 2) are the most studied
models.
aldehyde and 3-ethyl-4-methyl-1,5-dihydropyrrol-2-one were
dissolved in DMSO, and the solution was sparged with
nitrogen. Then a solution of KOH in water was added, and the
solution kept overnight at 60 °C (see the Experimental
Section). Moreover, we have synthesized a novel family of
benzalpyrrolinones with a methyl group instead of a hydrogen
atom in the vinylic position (compounds 2a−c). The
experimental procedure was similar to the one described
above, but the aldehyde is replaced by a ketone (see Scheme
1B).
In most of the synthesized compounds, just one isomer was
obtained in the reaction crude. The configuration of the central
carbon−carbon double bond of the most thermally stable
isomer was elucidated by NOE experiments (see the
Supporting Information). For compounds 1a−j, the Z isomer
is the thermally stable isomer, similar to other benzalpyrroli-
none analogues,43,44 whereas the E isomer is the most thermally
stable for compounds 2a,b, and a mixture of both isomers was
obtained in the synthesis of compound 2c (see Figure 3).
Hence, the replacement of the vinylic hydrogen by a methyl
group has a clear and direct effect on the thermal stability.
As mentioned in the Introduction, the presence of hydrogen-
bond interactions is crucial for these systems, controlling the
biological activity and photoisomerization degree. Therefore,
we have studied the formation and strength of hydrogen-bond
Figure 2. Structures of (A) dipyrrinone and (B) benzalpyrrolinone
derivatives.
Thus, while the biological relevance of phytochrome and
bilirubin is well-known and their biochemical features broadly
studied, more information is still needed to better understand
the basic functioning of these systems. With this aim, we
focused on the rationalization of the structural parameters that
can modify several relevant properties. First, we studied the
thermal properties of benzalpyrrolinone analogues and
proposed a novel model system where the vinylic hydrogen
has been replaced by a methyl group. Moreover, as the
formation of hydrogen-bond interactions in bilin pigments
plays an important role in phototherapy, we have evaluated the
B
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analyzed the amide proton chemical shift variation from CDCl3
to DMSO-d6 (Δδ) and its temperature dependence (Δδ/ΔT)
(see Table 1 and the Supporting Information). As can be
observed in Table 1, the chemical shift of the amide proton is
almost invariant with the solvent for compound 1c, whereas for
compounds 1f and 1g it changes 1.30 and 2.12 ppm,
respectively. Then, the temperature dependence, Δδ/ΔT, of
the amide proton chemical shift was analyzed. It is 1.7, 6.5, and
7.4 ppb/K for compounds 1c, 1f, and 1g, respectively (see
Table 1). These data reveal that the hydrogen bond of 1c is
strong (Δδ ≤ 0.2, Δδ/ΔT < 3) and those of 1f and 1g are weak
or nonexistent (Δδ > 0.2, Δδ/ΔT > 3).46
Hence, we can conclude that the thermal properties of these
model systems can be modified by replacing the vinylic
hydrogen by a methyl group and/or changing the aryl moiety.
Photochemical Study. Once the thermal properties have
been established, some photochemical properties were studied
such as the maximum absorption wavelength (λmax) and the
photostationary state (PSS) (Table 2). First, the absorption
Figure 3. X-ray structure of compound E-2c with thermal ellipsoids
shown at 50% probability.
interactions for compounds 1a−j. According to this thermal
property, derivatives 1a−j can be classified into three groups:
(i) monomers without hydrogen bond, (ii) monomers with an
intramolecular hydrogen bond, and (iii) dimers with an
intermolecular hydrogen bond.
Table 2. Maximum Absorption Wavelength (λmax), Molar
Absorptivity (ε), and Photostationary State (PSS) of
Compounds 1a−j and 2a−c in CHCl3
compd
λmax (nm)
ε (M−1 cm−1
)
PSS (Z/E)
In order to stablish the monomer/dimer structure and the
1
possibility of hydrogen-bond interaction, H NMR, DOESY,
Z-1a
Z-1b
Z-1c
Z-1d
Z-1e
Z-1f
Z-1g
Z-1h
Z-1i
Z-1j
E-2a
E-2b
E-2c
Z-2c
326
336
350
318
369
350
349
338
336
324
295
280
292
344
17200
20000
21500
22000
11000
26000
25000
19900
23300
20500
22800
16500
15000
34500
57:43
68:32
100:0
57:43
90:10
80:20
70:30
67:33
62:38
55:45
50:50
50:50
71:29
71:29
and X-ray data were recorded and analyzed (see the Supporting
Information). Compounds 1a,b,h−j are monomers that cannot
lead to hydrogen-bond interactions (group i) due to the lack of
NH or OH groups in the aryl moiety. In the case of compound
1d (4-Py), although an intermolecular hydrogen-bond
interaction could be possible, the X-ray structure (CCDC-
1
1457874, see Figure S2A) and the H NMR chemical shift of
the amide proton (δNH CDCl3 = 8.82 ppm, see the Supporting
Information) discard this hypothesis (at the concentration used
for the characterization).
It has been previously reported that compound 1e (Ar =
pyrrole) is a dimer due to intermolecular hydrogen-bond
interaction (group ii)45 and that compounds 1c,f,g present an
intramolecular hydrogen bond (group iii).43 However, the
strength of the hydrogen-bond interactions in compounds
1c,f,g has never been analyzed, and it is crucial in order to
understand both the thermal and photochemical properties. To
do this, the monomer vs dimer structure was checked for
compounds 1c,f,g by DOSY experiments (see the Supporting
Information) due to their similar structure compared to 1e. The
hydrodynamic volume ratio, compared to the pyrrole derivative
(1e), is ca. 2 (see Table 1), indicating that compounds 1c,f,g
are monomers.
spectrum for each compound was recorded (see Figure S1) and
the effect of the aryl substitution and hydrogen bond
interaction analyzed. For compounds 1b,c,e,f−i, a red-shift of
λmax was observed, compared to 1a, due to (i) introduction of
an electron-donating group in the aryl moiety (e.g., OMe in
1h,j), (ii) conjugation increase (e.g., a naphthyl ring in 1b), (iii)
five-membered ring aromaticity and electron confinement
reduction (e.g., pyrrole, furan, or thiophene rings in 1e,f,g
compared to the phenyl ring, 1a), and (iv) hydrogen bond
interactions (e.g., 1c,e). It is noteworthy that the shift observed
for 1e is surprisingly large, and it can be attributed not only to
the larger electronic density of the ring, compared to the furan
or thiophene rings, but also to its intermolecular hydrogen-
bond interaction, as previously concluded.
In order to further analyze the effect of the hydrogen-bond
interaction on λmax, we compared the values of Z-1c (2-Py)
with Z-1d (4-Py) and E-2c with Z-2c. For Z-1c and Z-2c, a red-
shift of 42 and 52 nm was observed due to their strong
hydrogen-bond interaction compared to Z-1d and E-2c,
respectively (see Figure 4).
Once the monomer structure of compounds 1c,f,g was set,
we evaluated their ability to establish intramolecular hydrogen-
bond interaction and their strength. With this goal in mind, we
Table 1. Hydrodynamic Volume (VH), Volume Ratio, Amide
Proton Chemical Shift Variation from CDCl3 to DMSO-d6
(Δδ), and Its Temperature Dependence (Δδ/ΔT) for
Compounds 1c,e−g
compd
VH (Å3)
vol ratio
Δδ (ppm)
Δδ/ΔT (ppb/K)
1e
1c
1f
197.07
97.15
88.14
95.54
1
2.03
2.24
2.06
0.18
1.30
2.12
1.7
6.5
7.4
Moreover, we have noticed that the absorption spectra of E-
1a and E-2a are blue-shifted compared to their respective Z
1g
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established, and two different external inputs, light and acid
(trifluoroacetic acid, TFA)/base (triethylamine, NEt3), to build
a cycle of four stages (see Figure 6).
1
In order to monitor the process, we first recorded the H
NMR spectrum of compound Z-1e, stage 1 in Figure 7. We
then added 1 equiv of TFA and observed that the signals shifted
to low fields, stage 2 in Figure 7. Once compound Z-1e is
protonated, the intermolecular hydrogen bond interaction is
disrupted and the system isomerizes after irradiation (PSS of
62/38 (Z/E)), stage 3 in Figure 7. Afterward, 1 equiv of NEt3
was added to again deprotonate the molecule, without
modifying the PSS, stage 4 in Figure 7. We observed how
the signals were shifted to high fields and one pair was
coincident with the ones of the parent compound. After
irradiation, the PSS was reached (see Table 2), observing that it
is enriched in the Z isomer (90%), stage 5 in Figure 7. This fact
can be explained by intermolecular hydrogen bonds in the Z
isomer that block the Z → E photoisomerization.
Figure 4. Normalized absorption spectra of Z-2c and E-2c.
isomers (see Figure 5). This difference can be explained in
terms of electronic structure (see the Discussion).
Photoisomerization Dynamics. In order to understand
the dynamic processes that take place after photoexcitation of
these model systems, we applied UV−vis transient absorption
spectroscopy (see the Experimental Section for details). We
selected compounds Z-1a and E-2a as the representative
derivatives for each model family and CHCl3 as solvent. By
comparing the performance of these compounds, we could
understand the effect of the critical structural modifications
discussed above. The effect of the hydrogen bonding, while
relevant for the photochemical properties as shown in the
previous sections, would only affect the time scale of the
excited-state decay (see below) and, thus, will not be
considered here. The excitation wavelengths were chosen to
be 340 nm for Z-1a and 300 nm for E-2a and thus slightly red-
shifted with respect to the absorption maxima (see Table 2 and
Figure 5) in order to reduce excess energy in the excited state.
Additionally, experiments were performed at 340 nm for E-2a
in order to compare the relaxation behavior of both compounds
excited with the same energy. Figure 8 shows the transient
absorption changes (ΔOD) mapped as a function of the probe
wavelengths and pump−probe time delay, where positive ΔOD
is coded in red.
After studying the absorption properties, all compounds were
irradiated in a 400 W medium-pressure Hg lamp in a Pyrex
NMR tube in order to measure their photostationary state
(PSS). The photoisomerization was easily followed by 1H
NMR as the chemical shifts of certain protons of both isomers
are different enough (see Figure S6). Irradiation was kept until
the PSS was reached (Table 2). In this case, no significant
variation due to the electronic nature of the substituent or the
aryl moiety was observed (see compounds 1b,d,h-j,2b in Table
2). Whereas, the hydrogen bond interaction, either intra- or
intermolecular, influences the photoisomerization process (see
compounds 1c,e-g,2c in Table 2). For compound 1c (2-Py),
where a strong hydrogen bond is formed, the photoisomeriza-
tion is completely blocked. Along this line, the PSS measured
for 1e (pyrrole), 1f (furan), and 1g (thiophene) is lower than
expected, and the percentage of photoisomer, E isomer,
increases from 1e < 1f < 1g as the intramolecular hydrogen
bond strength decreases in the same order (see Synthesis and
Thermal Properties). Similarly, the PSS of 2c is enriched in Z
isomer as an intramolecular hydrogen bond interaction is
possible but not strong enough to block the photoisomeriza-
tion. Hence, we can conclude that the main feature affecting the
photochemical properties (λmax and PSS) is the hydrogen-bond
interaction.
Regarding compound Z-1a, a positive band centered at 490
nm was observed (see Figure 8A) that corresponds to the
excited-state absorption (ESA). For compound E-2a, two
positive absorption bands around 520 and 660 nm were
observed (see Figure 8B,C). The transient absorption profiles
pumped at 340 nm were globally fitted by a biexponential decay
revealing two ultrafast time constants on the femtosecond (τ1)
The direct dependence of the photoisomerization extent on
the hydrogen bond strength can be also useful in order to
control the photoisomerization process by means of different
external inputs. With this aim, we selected compound Z-1e,
whose intermolecular hydrogen-bond interactions are well
Figure 5. Normalized absorption spectra of compounds (A) 1a and (B) 2a.
D
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Figure 6. Four-stages cycle for compound 1e using two different external inputs.
Table 3. Time Constants Obtained from Global Fits of Each
Transient Absorption Spectrum for Compounds 1a and 2a
compd
λpump (nm)
τ1 (ps)
τ2 (ps)
1a
2a
2a
340
340
300
0.18
0.26
1.87
0.97
0.61
Concerning Figure 5, the photochemical isomers which are
built after excitation reveal absorption bands in the ultraviolet
region. Since the sapphire-generated white light is limited to
435 nm, single-color probe experiments were additionally
performed in the UV to check for complete ground-state
recovery. The corresponding transients of Figure 9 show
photoproduct absorption (PA) as well as ground-state
bleaching (GSB) of the photochemical and the most thermally
stable isomers for compound Z-1a pumped at 340 nm and
compound E-2a pumped at 300 nm, respectively.
1
Figure 7. H NMR signals in the aromatic region for the four stages
needed to complete the cycle.
Regarding compound Z-1a, 345 and 330 nm were selected as
the probe wavelengths. Their transient absorption profiles are
quite similar (see Figure 9A). An intense negative signal is
observed mainly due to the GSB of the thermally stable isomer.
At 330 nm, a positive band is observed around time zero
indicating ESA that dominates the visible spectral part (see
Figure 9A). However, in the UV part, ESA is superimposed by
GSB, which dominates in this regime and can be attributed to
pure ground-state recovery. At 345 nm, the early-time ESA is
not seen, most likely due to chirp under nonoptimized
dispersion compensation conditions. The slightly different
recovery profiles at 330 and 345 nm on a picosecond time scale
may arise from absorption of hot vibrational photoproduct or
reactant (HGSA). This superposition is much more obvious for
E-2a (see below). At longer times, the transients do not fully
recover, indicating slower processes and/or incomplete switch-
ing. This is in line with Figure 5, demonstrating that the E and
Z isomers show significant absorptive overlap which cannot be
completely separated by photoexcitation.
Compound E-2a was pumped at 300 nm and probed at 345,
330, and 305 nm; see Figure 9B. As a consequence of the
observed superposition of excited-state absorption and ground-
state bleaching in the UV region, these transient absorption
profiles cannot be described by simple exponential kinetics. A
detailed discussion on how the individual components would
be appropriately treated is given by earlier studies of Briand and
Temps et al.47−50 In our case, such a thorough analysis is not
possible because the important spectral window between 345
and 430 nm where most likely hot ground-state absorption
prevails is lacking. On the other hand, a detailed analysis of
these data is well beyond the scope of this paper.
Figure 8. Transient absorption changes (ΔmOD) as a function of the
probe wavelength and pump−probe time delay pumped at 340 nm of
(A) Z-1a, (B) E-2a, and (C) E-2a pumped at 300 nm.
and (sub-) picosecond (τ2) time scale. The results are quite
similar for compound Z-1a and E-2a. In contrast, the transient
absorption profile of E-2a pumped at 300 nm was modeled by a
monoexponential decay (τ2). All time constants are listed in
Table 3. However, as τ1 is close to the experimental time
resolution, the corresponding decay could also be faster than
the values may suggest.
Computational Study. Finally, a computational study of
compounds 1a, 1c, 1f, and 2a has been performed. The
different Z/E relative stabilities and excited-state lifetimes of 1a
E
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Figure 9. Transient absorption profiles at selected probe wavelengths for (A) compound Z-1a, pumping at 340 nm, and (B) compound E-2a,
pumping at 300 nm.
and 2a and, moreover, the effect of hydrogen bonding on the
excited state compared to the MEPs of 1a, 1c, and 1f have been
analyzed. Regarding the different thermal behavior of 1a and
2a, first the ground-state minima of Z-1a, E-1a, Z-2a, and E-2a
were optimized at the MP2/6-31g(d) level of theory, taking
into account the solvent effect (see Computational Details).
Concerning the thermal stability, it was found that Z-1a is 3.5
kcal·mol−1 more stable than E-1a, whereas E-2a is around 1
kcal·mol−1 more stable than Z-2a. The inversion of the relative
thermal stability for compound 2a can be explained by the
steric hindrance between the two methyl groups in Z-2a
(Figure 10C), which is relieved in E-2a (Figure 10D) by
Table 4. Structural Parameters (φCZ, φCE, φNZ, φNE, and
d
CC) for the Ground-State Minima of Compounds Z-1a, E-
1a, Z-2a, and E-2a Calculated at the MP2/6-31g(d) Level of
theory
compd
dCC (Å)
φCZ, φCE (deg)
φNZ, φNE (deg)
Z-1a
E-1a
Z-2a
E-2a
1.357
1.357
1.364
1.362
−4.5
12.6
−3.3
15.0
−1.5
5.2
−5.8
3.3
account the solvent effect (see the Computational Details). For
all of the compounds, the optical bright state is the first singlet
excited state, S1, whose electronic transition is mainly described
by the excitation of one electron from the bonding, π, to the
antibonding, π*, orbital of the central carbon−carbon double
bond (see Figure S7). This means that after excitation the
photoisomerizable formal double bond is elongated, allowing
rotation around this bond. The calculated excitation energies
(in nm) were 348 (317) and 310 (284) for compounds Z-1a
(E-1a) and Z-2a (E-2a), respectively (Table S3), in very good
agreement with the absolute experimental values as well as the
blue/red-shift experimentally observed.
Furthermore, the minimum energy paths (MEPs) of
compounds Z-1a and E-2a were computed. From the
Franck−Condon (FC) geometry, both Z-1a and E-2a minimize
the energy in S1 along the stretching coordinate. The formal
carbon−carbon double bond is elongated, resulting in a bond
length alternation of single and double bonds, as predicted by
the electronic nature of the vertical transition. Once the
stretching mode is relaxed, the system starts to rotate around
the photoisomerizable central carbon−carbon double bond
until it reaches a conical intersection (CI) with the ground state
at ca. 90° of rotation (see Figure 11). From here, the system
may decay to the ground state, completing the photo-
isomerization or reverting to the initial isomer. Analyzing the
data in Figure 11, we observe that the minimum energy path
along the torsion coordinate, (φNZ or φNE) for Z-1a is very flat,
whereas for compound E-2a the path is much steeper and the
excess vibrational energy from the FC to the CI is ca. 14 kcal·
mol−1 higher, which could accelerate the motion toward the CI.
Finally, the minimum energy paths of Z-1c and Z-1f have
been computed and compared with the one of Z-1a (see Figure
11 and Figure S9). As previously shown (see Synthesis and
Thermal Properties), a hydrogen-bond interaction is present in
compounds Z-1c (Ar = 2-pyridine) and Z-1f (Ar = 2-furan)
Figure 10. Ground-state minima structures of (A) Z-1a, (B) E-1a, (C)
Z-2a, and (D) E-2a. φCZ, φCE, φNZ, φNE, φPh, and dCC are depicted.
rotation of the phenyl ring (ca. 60°). For compound 1a, the Z
isomer is the most thermally stable due to its more planar and
conjugated structure compared to E-1a (φCZ(CE) = −4.5
(12.6)°; φPh = 37 (57)° for Z-1a (E-1a)). Moreover, analyzing
the values of φCE, φCZ, and dCC (Table 4), it is observed that
the carbon−carbon double-bond distance is slightly longer for
2a, compared to 1a, and that the torsion around this bond is
more advanced for the E isomers.
The absorption spectra and minimum energy paths of these
two compounds were then calculated to understand the
different excited-state dynamics experimentally found by TAS.
First, the absorption spectra of Z-1a, E-1a, Z-2a, and E-2a were
simulated at the TD-DFT/6-31g(d) level of theory, taking into
F
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Article
rotation of the phenyl ring (see Figure 3). Hence, we may
control the thermal carbon−carbon double bond configuration
by means of minor structural changes.
In addition, the establishment of hydrogen-bond interactions
is a crucial feature in bilirubin and bilin pigments. They do
influence not only the thermal properties but also the
photochemical ones such as the absorption spectrum or the
photoisomerization process. For instance, a significant red-shift
of the absorption spectrum is observed for the isomer with
hydrogen bonds which, in turn, increases the separation
between the absorption bands of both isomers (Figure 5).
We can take advantage of this evidence and guide the design of
derivatives with long absorption wavelength, desirable for
biological applications, and large absorption difference of both
isomers, increasing the irradiation selectivity. On the other
hand, we have confirmed that the strength of the hydrogen
bond has a direct effect on the photoisomerization extent, more
in particular on the PSS. The percentage of the initial isomer in
the PSS can be modulated by the hydrogen-bond strength, even
blocking the photoisomerization process as observed for
compound 1c (Table 2).
Finally, we can give insight into the first steps and the
photoisomerization mechanism of these model systems by a
combined experimental TAS and theoretical analysis. Consid-
ering the transient absorption spectra pumped at 340 nm
(Figure 8A,B), two time constants were obtained characterizing
the excited-state dynamics of Z-1a and E-2a (Table 3).
Analyzing the MEPs computed for our models, the first time
constant, τ1, is attributable to the activation of the stretching
mode, high frequency mode, which elongates the central
carbon−carbon double bond from the FC region (see Figure
11 and Figure S8). This process takes place within a few
hundred femtoseconds. Compared to the literature, such an
ultrafast component was also observed in phytochrome.51 As
observed in the MEPs (Figure 11), once the stretching motions
are completed, the system minimizes the energy along the
torsional mode (low frequency mode), rotating around the
former carbon−carbon double bond upon reaching a conical
intersection with the ground state. This relaxation pathway is
associated with the second time constant, τ2, on a (sub)-
picosecond time scale (see Figure 12).
Figure 11. Minimum energy paths of Z-1a, Z-1f, and E-2a along the
torsion coordinate (φNZ and φNE, respectively) in S1 from the FC
structure.
that is strong enough to block the photoisomerization process
for 1c. By analyzing their MEPs, we can observe that after
excitation to the bright state the system first activates the
stretching mode, similarly to Z-1a. Then, a minimum in S1 has
been found for both Z-1c and Z-1d, due to the hydrogen-bond
interaction that hampers the rotation around the photo-
isomerizable double bond. From here, the system has to
overcome an energy barrier along the torsion coordinate up to
reaching the S1/S0 conical intersection. The energy barriers
computed were 3.5 and 4.6 kcal/mol for Z-1f and Z-1c, in line
with the stronger hydrogen-bond interaction experimentally
measured for Z-1c (see Figure S9). Hence, it can be concluded
that the presence of a hydrogen-bond interaction clearly
influences the excited state, increasing the energy barrier along
the torsion coordinate which value is directly related to the
hydrogen bond strength. For the same reason, it can be stated
that the excited-state lifetime will be increased with the
hydrogen-bond strength.
DISCUSSION
■
First, the rationalization of the structural effects that control the
Z/E relative thermal stability is a key point to facilitate the
isolation and characterization of the photoproduct and, even
more relevant, to decrease the dark reversion rate, as discussed
in the Introduction. With this purpose, we analyzed three cases:
(i) compound 1a, whose thermally stable isomer is the Z
isomer; (ii) compound 2a, for which the Z/E relative stability is
reverted and the E isomer is the most thermally stable, and (iii)
compound 2c, an intermediate situation where the energy of
both isomers is similar. To understand these differences, we can
argue in terms of steric hindrance, conjugation extent, and
hydrogen-bond interactions. Regarding compound 1a, the Z
isomer is the most thermally stable due to its more planar and,
hence, more conjugated structure, compared to E-1a, as
suggested by the structural parameters of the ground-state
minima computed (Table 4), whereas for 2a the main effect
responsible for the Z/E relative stability inversion is the steric
hindrance between the two methyl groups in the Z isomer,
destabilizing it (see Figure 10C). Moreover, the similar thermal
stability found for E-2c and Z-2c can be easily explained: the Z
isomer is destabilized by steric hindrance between the two
methyl groups of the structure but stabilized by intramolecular
hydrogen-bond interaction (δamide proton= 11.44 ppm, see the
Supporting Information). On the other hand, the E isomer is of
comparable stability as the steric hindrance is released by
Moreover, no intermediates such as minima in the excited
state were found along the MEP. This evidence is in line with
the time scale of τ2, as it is generally larger when intermediates
are present in the process (e.g., in phytochrome the first
Figure 12. Schematic representation of the photoisomerization
process for compounds Z-1a and E-2a.
G
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Article
isomerized excited state intermediate is formed in 3 ps, as there
is a local minimum due to a interaction of a ring with the
protein).51 However, the excited-state decay of these models is
longer lived than the ones found for downhill processes such
the photoisomerization of the retinal protonated Schiff base in
rhodopsin (∼200 fs).52 This fact can be due to the flat profile
along the torsion coordinate in the MEP that slows the process.
Additionally, we can compare the photodynamics of
compounds Z-1a and E-2a. We observed that the excited
state decay, τ2, is almost two times faster for E-2a when
pumping at 340 nm. This fact is in agreement with the steeper
path along the torsion coordinate computed for E-2a. The
different potential energy surface topologies can be rationalized
in terms of structural parameters related to the photo-
isomerization process. Along this line, we can analyze the
central carbon−carbon double-bond distance (dCC) and its
pretwist (φCZ, φCE). Concerning dCC, it is slightly larger for E-
2a (Table 4) compared to Z-1a in the ground-state minima,
and this trend is kept along the MEP (see Figure S4). This
implies a higher character of single bond of the photo-
isomerizable double bond for E-2a which could favor the
rotation around this bond. Another important structural
parameter that could influence the photochemistry is the
pretwist of the photoisomerizable double bond. The pretwist of
strength. Hence, the design of derivatives with hydrogen bonds
could be used to stabilize to a large extent one of the isomers
and red-shift the λmax but it can also decrease the photo-
isomerization efficiency.
In addition, the excited-state dynamics of Z-1a and E-2a have
been studied by TAS and the MEPs of Z-1a, Z-1c, Z-1f, and E-
2a computed along S1. Regarding Z-1a and E-2a, a fast
relaxation out of the FC region due to stretching motions was
observed, τ1, followed by a barrierless rotation around the
former central double bond, in agreement with the time scale of
τ2. Moreover, we observed that the excited-state decay of E-2a
is sped up regarding to Z-1a, due mainly to their different
electronic structure (dCC and pretwist). Finally, it has been
shown that the hydrogen-bond interaction has a clear influence
on the MEP, appearing as an energy barrier along the torsion
coordinate whose value depends on the interaction strength.
In summary, this study can guide the design of both
structurally modified phytochrome chromophores and novel
protein mutants.53,54 This may be relevant for the tuning of the
properties of phytochromes and also to generate novel
chromoproteins with different functions. Moreover, this work
can help to better understand the photodynamics of the
chromophores in vivo.
E-2a (φCE = 15.0°) is larger than the one of Z-1a (φCZ
=
EXPERIMENTAL SECTION
■
−4.5°). This increases the initial velocity out of the FC region
along the torsion coordinate for E-2a, finding a steepest profile.
Thus, the electronic and steric effects of the methyl group in
the vinylic position modify both the relative stability of Z/E
isomers and their photochemistry.
General Information. 1H and 13C NMR spectra were recorded on
a 400 MHz NMR spectrometer. Signal patterns are depicted as s
(singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).
Electrospray mass spectra were recorded on a mass spectrometer using
the ESI mode. For the photostationary state measurement, a solution
of the sample was placed in a Pyrex NMR tube and irradiated in a 400
W medium-pressure mercury lamp, and the photoproduct formation
was quantified by NMR. X-ray data were collected with an area-
detector diffractometer using graphite-monochromatic Mo Kα
irradiation. Images were processed using the DENZO and
SCALEPACK suite of programs.55
Considering the transient absorption spectrum of E-2a
pumped at 300 nm (Figure 8C), monoexponential decay of
ΔmOD was observed within 0.61 ps (τ2, Table 3). In contrast
to the experiments performed at 340 nm, excitation at 300 nm
leads to higher excess energy in the initially excited state. Thus,
it is plausible that the activation of the stretching mode is
within our experimental time resolution. The obtained time
constant, τ2, is associated with the excited-state dynamics
toward a conical intersection with the ground state.
Finally, we have analyzed the effect of the hydrogen-bond
interaction in the excited state by comparing the MEPs of Z-1a,
Z-1c, and Z-1f. It has been demonstrated that a minimum in S1
appears for 1c and 1f as a consequence of the hydrogen-bond
interaction. Moreover, the system has to overcome an energy
barrier along the torsion coordinate proportional to the
hydrogen-bond strength. Hence, the stronger is the interaction
the higher is the energy barrier, increasing the excited state
lifetime and even, blocking the photoisomerization process as is
the case of Z-1c.
Time-Resolved Experimental Methods. The ultrafast dynamics
of the synthesized compounds 1a and 2a were studied by femtosecond
transient absorption spectroscopy. Pulses of 150 fs duration with 1.6
mJ/pulse at a repetition rate of 1 kHz and a central wavelength of 775
nm were generated by a CPA 2210 laser system.56 The experimental
setup employed for transient absorption spectroscopy is described in
more detail elsewhere.57 The pump wavelengths of 300 and 340 nm
were generated by frequency doubling. For probing, a sapphire
generated white light in the spectral range from 435 to 680 nm. The
experimental setup employed for single-color probe measurements is
described in more detail elsewhere.58 The pump wavelengths of 300
and 340 nm were generated by sum frequency mixing.59 The probe
wavelength of 500 nm was generated by a noncollinearly optical
parametric amplifier (NOPA) system.60 Additionally, probe wave-
lengths of 305, 330, and 345 nm were generated by frequency
doubling of the visible NOPA output. All measurements were carried
out under magic angle (54.7°) conditions at room temperature and an
optical density of 2.5 in order to minimize the group velocity
mismatch of pump and probe beam. Moreover, to avoid the local
buildup of photoproducts, the sample was continuously pumped
through a fused silica cuvette with 1 mm optical path length. Data
acquisition was performed using in house software in a Labview
environment resulting in pump-induced, time-resolved changes of the
optical density (ΔOD). For details, see ref 57. The error is estimated
to be 0.2 ps for τ1 and 0.1 ps for τ2.
CONCLUSION
■
In this work, we have presented a combined experimental
(synthesis and characterization) and theoretical study of two
different families of bilin chromophores and bilirubin model
systems. First, we have been able to control the Z/E relative
thermal stability by minor structural changes. Thanks to the
analysis of the structural parameters and the NMR data, we
have concluded that the steric hindrance, conjugation extent
and hydrogen bond interactions are the principal factors that
govern this property. In addition, we have confirmed that
hydrogen bond interactions influence also the photochemical
properties, red-shifting the absorption spectrum, or modifying
the PSS and the MEP according to the hydrogen bond
Computational Details. Ground-state minima were optimized at
the MP2/6-31g(d) level of theory taking into account the solvent
effect by means of the polarizable continuum model (PCM).61 The
absorption spectra and the minimum energy paths on the excited state
were simulated at the TD-DFT/6-31g(d) level of theory with the
CAM-B3LYP62 hybrid functional, taking into consideration the solvent
H
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Article
effect with the PCM model. All calculations were performed with
Gaussian 09 software.63
(Z)-3-Ethyl-5-((furan-2-yl)methylene)-4-methyl-1H-pyrrol-2(5H)-
1
one (Z-1f). Yield = 80% (0.1625 g, mp: 97−98 °C). H NMR (400
MHz, CDCl3): δ 8.26 (s, 1H), 7.45 (s, 1H), 6.45−6.44 (m, 1H), 6.40
(d, J = 3.4 Hz, 1H), 5.85 (s, 1H), 2.37 (q, J = 7.6 Hz, 2H), 2.05 (s,
3H), 1.11 (t, J = 7.6 Hz, 3H). 13C NMR (400 MHz, CDCl3): δ 171.6
(s), 151.9 (s), 143.2 (s), 139.9 (s), 136.0 (s), 133.8 (s), 112.1 (s),
111.3 (s), 95.3 (s), 16.9 (s), 13.4 (s), 9.5 (s). UV−vis (CHCl3): λ(nm)
= 220 (ε = 6300 M−1 cm−1), 283 (ε = 11000 M−1 cm−1), 343 (ε =
26000 M−1 cm−1). EM-ES(+): (C12H13NO2 + H) calcd 204.1024,
found 204.1028. Observations: dark reddish solid.
Synthesis of Compounds 1a−j and 2a−c. In a round-bottom
flask were dissolved 1 mmol of the desired carbonyl compound and 2
mmol (0.250 g) of 3-ethyl-4-methyl-1H-pyrrol-2(5H)-one in 5 mL of
DMSO. Once those compounds were dissolved, 1.5 mL of KOH 4 M
was added. The mixture was stirred under argon at 60 °C for 12 h. The
reaction mixture was poured into 20 mL of water, and the precipitate
obtained was collected by vacuum filtration and washed with cold
water.
(Z)-3-Ethyl-4-methyl-5-((thiophene-2-yl)methylene)-1H-pyrrol-
2(5H)-one (Z-1g). Yield = 98% (0.2147 g). 1H NMR (400 MHz,
CDCl3): δ 7.57 (s, 1H), 7.37 (d, J = 5.0 Hz, 1H), 7.12 (d, J = 3.4 Hz,
1H), 7.08 (t, J = 4.2 Hz, 1H), 6.27 (s, 1H), 2.39 (q, J = 7.6 Hz, 2H),
2.10 (s, 3H), 1.13 (t, J = 7.6 Hz, 3H). 13C NMR (400 MHz, CDCl3): δ
171.8 (s), 141.1 (s), 138.3 (s), 136.3 (s), 133.9 (s), 128.1 (s), 127.9
(s), 126.4 (s), 101.1 (s), 17.0 (s), 13.3 (s), 9.6 (s). UV−vis (CHCl3):
λ(nm) = 202 (ε = 14700 M−1 cm−1), 234 (ε = 4800 M−1 cm−1), 350
(ε = 25000 M−1 cm−1). EM-ES(+): (C12H13NOS + H) calcd
220.0796, found 220.0787. Observations: yellow solid.
(Z)-5-Benzylidene-3-ethyl-4-methyl-1H-pyrrol-2(5H)-one (Z-1a).
Yield = 81% (0.1726 g, mp 136−137 °C). 1H NMR (400 MHz,
CDCl3): δ 7.89 (s, 1H), 7.43 (d, J = 4.4 Hz, 4H), 7.35−7.30 (m, 1H),
6.13 (s, 1H), 2.43 (q, J = 7.6 Hz, 2H), 2.16 (s, 3H), 1.17 (t, J = 7.6 Hz,
3H). 13C NMR (400 MHz, CDCl3): δ 172.7 (s), 141.2 (s), 138.1 (s),
135.2 (s), 133.9 (s), 129.1 (s), 128.5 (s), 127.7 (s), 108.0 (s), 16.9 (s),
13.3 (s), 9.7 (s). UV−vis (CHCl3): λ(nm) = 196 (ε = 25500 M−1
cm−1), 228 (ε = 6800 M−1 cm−1), 326 (ε = 17200 M−1 cm−1). EM-
ES(+): (C14H15NO + H) calcd 214.1232, found 214.1233.
Observations: yellow solid.
(Z)-5-(4-Methoxybenzylidene)-3-ethyl-4-methyl-1H-pyrrol-2(5H)-
one (Z-1h). Yield = 83% (0.2018 g, mp 107−108 °C). 1H NMR (400
MHz, CDCl3): δ 7.69 (s, 1H), 7.33 (d, J = 8.6 Hz, 2H), 6.93 (d, J = 8.6
Hz, 2H), 6.04 (d, J = 6.6 Hz, 1H), 3.84 (s, 3H), 2.39 (q, J = 7.6 Hz,
2H), 2.11 (s, 3H), 1.13 (t, J = 7.6 Hz, 3H). 13C NMR (400 MHz,
CDCl3): δ 172.7 (s), 159.2 (s), 141.3 (s), 136.7 (s), 133.3 (s), 129.9
(s), 127.8 (s), 114.7 (s), 108.0 (s), 55.4 (s), 16.9 (s), 13.4 (s), 9.7 (s).
UV−vis (CHCl3): λ(nm) = 191 (ε = 29700 M−1 cm−1), 240 (ε = 9200
M−1 cm−1), 338 (ε = 19900 M−1 cm−1). EM-ES(+): (C15H17NO2 +
H) calcd 244.1337, found 244.1344. Observations: yellow solid.
(Z)-5-(2-Methoxybenzylidene)-3-ethyl-4-methyl-1H-pyrrol-2(5H)-
(E)-5-Benzylidene-3-ethyl-4-methyl-1H-pyrrol-2(5H)-one (E-1a).
1H NMR (400 MHz, CDCl3) δ 9.07 (s, 1H), 7.24−7.36 (m, 5H),
6.62 (s, 1H), 2.35 (q, J = 7.3 Hz, 1H), 1.73 (s, 1H), 1.09 (t, J = 7.5 Hz,
1H). 13C NMR (400 MHz, CDCl3): δ 172.0 (s), 139.0 (s), 139.0 (s),
138.2 (s), 135.2 (s), 129.7 (s), 128.1 (s), 127.6 (s), 113.9 (s), 16.7 (s),
13.3 (s), 13.2 (s). UV−vis (CHCl3): λ(nm) 304 (ε = 13800 M−1
cm−1). EM-ES(+): (C14H15NO + H) calcd 214.1232, found 214.1233.
(Z)-3-Ethyl-4-methyl-5-((naphthalen-2-yl)methylene)-1H-pyrrol-
2(5H)-one (Z-1b). Yield = 88% (0.2316 g, mp 191−192 °C). 1H NMR
(400 MHz, CDCl3): δ 8.14 (s, 1H), 7.92 (s, 1H), 7.89−7.80 (m, 3H),
7.54−7.44 (m, 3H), 6.24 (s, 1H), 2.42 (q, J = 7.6 Hz, 2H), 2.16 (s,
3H), 1.15 (t, J = 7.6 Hz, 3H). 13C NMR (400 MHz, CDCl3): δ 172.9
(s), 141.4 (s), 138.4 (s), 133.9 (s), 133.7 (s), 132.7 (s), 132.6 (s),
128.8 (s), 128.2 (s), 127.7 (s), 127.6 (s), 126.7 (s), 126.5 (s), 126.4
(s), 108.2 (s), 16.9 (s), 13.4 (s), 9.7 (s). UV−vis (CHCl3): λ(nm) =
213 (ε = 33400 M−1 cm−1), 279 (ε = 21400 M−1 cm−1), 336 (ε =
20000 M−1 cm−1). EM-ES(+): (C18H17NO + H) calcd 264.1388,
found 264.1387. Observations: yellow solid.
(Z)-3-Ethyl-4-methyl-5-((pyridin-2-yl)methylene)-1H-pyrrol-
2(5H)-one (Z-1c). Yield = 94% (0.2013 g). 1H NMR (400 MHz,
CDCl3): δ 10.48 (s, 1H), 8.57 (d, J = 4.7 Hz, 1H), 7.66 (t, J = 7.7 Hz,
1H), 7.25 (d, J = 7.9 Hz, 1H), 7.14−7.07 (m, 1H), 5.93 (s, 1H), 2.43
(q, J = 7.6 Hz, 2H), 2.12 (s, 3H), 1.16 (t, J = 7.6 Hz, 3H). 13C NMR
(400 MHz, CDCl3): δ 172.2 (s), 155.8 (s), 149.2 (s), 142.8 (s), 140.0
(s), 136.6 (s), 135.2 (s), 124.6 (s), 121.0 (s), 103.5 (s), 16.9 (s), 13.4
(s), 9.4 (s). UV−vis (CHCl3): λ(nm) = 192 (ε = 34300 M−1 cm−1),
279 (ε = 5600 M−1 cm−1), 350 (ε = 21500 M−1 cm−1). EM-ES(+):
(C13H14N2O + H) calcd 215.1184, found 215.1212. Observations:
yellow solid.
1
one (Z-1i). Yield = 89% (0.2164 g, mp 147−148 °C). H NMR (400
MHz, CDCl3): δ 7.85 (s, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.30 (t, J = 7.8
Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.26 (s,
1H), 3.90 (s, 3H), 2.41 (q, J = 7.5 Hz, 2H), 2.16 (s, 3H), 1.15 (t, J =
7.6 Hz, 3H). 13C NMR (400 MHz, CDCl3): δ 172.2 (s), 156.8 (s),
141.2 (s), 138.0 (s), 133.9 (s), 129.9 (s), 129.3 (s), 124.2 (s), 121.2
(s), 111.3 (s), 103.9 (s), 55.7 (s), 16.9 (s), 13.3 (s), 9.7 (s). UV−vis
(CHCl3): λ(nm) = 203 (ε = 34800 M−1 cm−1), 336 (ε = 23300 M−1
cm−1).EM-ES(+): (C15H17NO2 + H) calcd 244.1337, found 244.1334.
Observations: yellow solid.
(Z)-5-(4-Bromobenzylidene)-3-ethyl-4-methyl-1H-pyrrol-2(5H)-
1
one (Z-1j). Yield = 94% (0.2736 g, mp 146−147 °C). H NMR (400
MHz, CDCl3): δ 8.38 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4
Hz, 2H), 6.00 (s, 1H), 2.38 (q, J = 7.5 Hz, 2H), 2.11 (s, 3H), 1.13 (t, J
= 7.5 Hz, 3H). 13C NMR (400 MHz, CDCl3): δ 173.3 (s), 141.3 (s),
138.5 (s), 134.0 (s), 132.1 (s), 130.3 (s), 121.6 (s), 107.1 (s), 17.0 (s),
13.3 (s), 9.7 (s). UV−vis (CHCl3): λ(nm) = 194 (ε = 26200 M−1
cm−1), 233 (ε = 9800 M−1 cm−1), 324 (ε = 20500 M−1 cm−1). EM-
ES(+): (C14H14BrNO + H) calcd 292.0337, found 292.0332.
Observations: yellow solid.
(Z)-3-Ethyl-4-methyl-5-((pyridin-4-yl)methylene)-1H-pyrrol-
2(5H)-one (Z-1d). Yield = 76% (0.1627 g, mp 144−145 °C). 1H NMR
(400 MHz, CDCl3): δ 8.82 (s, 1H), 8.63 (d, J = 5.0 Hz, 2H), 7.36 (d, J
= 5.3 Hz, 2H), 5.99 (s, 1H), 2.43 (q, J = 7.5 Hz, 2H), 2.15 (s, 3H),
1.17 (t, J = 7.6 Hz, 3H). 13C NMR (400 MHz, CDCl3): δ 173.2 (s),
150.4 (s), 142.6 (s), 141.3 (s), 141.2 (s), 135.2 (s), 122.9 (s), 104.9
(s), 17.0 (s), 13.2 (s), 9.7 (s). UV−vis (CHCl3): λ(nm) = 197 (ε =
9500 M−1 cm−1), 223 (ε = 5100 M−1 cm−1), 318 (ε = 22000 M−1
cm−1). EM-ES(+): (C13H14N2O + H) calcd 215.1184, found
215.1188. Observations: yellow solid.
(Z)-5-((1H-Pyrrol-2-yl)methylene)-3-ethyl-4-methyl-1H-pyrrol-
2(5H)-one (Z-1e). Yield = 80% (0.1617 g, mp: 110−111 °C). 1H
NMR (400 MHz, CDCl3): δ 11.09 (s, 1H), 10.77 (s, 1H), 7.05 (s,
1H), 6.45 (s, 1H), 6.28 (s, 1H), 6.17 (s, 1H), 2.44 (q, J = 7.5 Hz, 2H),
2.13 (s, 3H), 1.18 (t, J = 7.5 Hz, 3H). 13C NMR (400 MHz, CDCl3): δ
174.1 (s), 142.1 (s), 130.8 (s), 130.7 (s), 127.5 (s), 123.3 (s), 115.9
(s), 109.9 (s), 103.3 (s), 17.0 (s), 13.4 (s), 9.7 (s). UV−vis (CHCl3):
λ(nm) = 199 (ε = 9500 M−1 cm−1), 265 (ε = 5700 M−1 cm−1), 364 (ε
= 11000 M−1 cm−1). EM-ES(+): (C12H14N2O + H) calcd 203.1184,
found 203.1184. Observations: green brown solid.
(E)-3-Ethyl-4-methyl-5-(1-phenylethylidene)-1H-pyrrol-2(5H)-one
1
(E-2a). Yield = 67% (0.1528 g, mp 200−201 °C). H NMR (400
MHz, CDCl3): δ 8.99 (s, 1H), 7.34 (t, J = 6.7 Hz, 3H), 7.22 (d, J = 7.7
Hz, 2H), 2.34−2.22 (m, 5H), 1.37 (s, 3H), 1.04 (t, J = 7.5 Hz, 3H).
13C NMR (400 MHz, CDCl3): δ 171.9 (s), 141.5 (s), 140.3 (s), 135.8
(s), 135.0 (s), 128.8 (s), 128.4 (s), 127.6 (s), 122.9 (s), 22.7 (s), 16.6
(s), 13.3 (s), 13.2 (s). UV−vis (CHCl3): λ(nm) = 241 (ε = 18900 M−1
cm−1), 295 (ε = 22800 M−1cm−1). EM-ES(+): (C15H17NO + H) calcd
228.1388, found 228.1383. Observations: yellow solid.
(Z)-3-Ethyl-4-methyl-5-(1-phenylethylidene)-1H-pyrrol-2(5H)-one
1
(Z-2a). H NMR (400 MHz, CDCl3): δ 7.42−7.22 (m, 1H), 6.90 (s,
1H), 2.41−2.33 (m, 2H), 2.30 (s, 1H), 1.09 (t, J = 7.5 Hz, 1H). 13C
NMR (400 MHz, CDCl3): δ 170.1 (s), 141.8 (s), 139.5 (s), 136.1 (s),
134.2 (s), 128.8 (s), 127.8 (s), 127.7 (s), 121.4 (s), 19.9 (s), 16.5 (s),
14.4 (s), 13.4 (s). UV−vis (CHCl3): λ(nm) = 306 (ε = 16600 M−1
cm−1). EM-ES(+): (C15H17NO + H) calcd 228.1388, found 228.1383.
(E)-3-Ethyl-4-methyl-5-(1-(naphthalen-3-yl)ethylidene)-1H-pyr-
1
rol-2(5H)-one (E-2b). Yield = 86% (0.2383 g, mp 147−148 °C). H
NMR (400 MHz, CDCl3): δ 9.68 (s, 1H), 7.89−7.81 (m, 3H), 7.69 (s,
1H), 7.54−7.49 (m, 2H), 7.37 (d, J = 8.4 Hz, 1H), 2.39 (s, 3H), 2.33
I
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(q, J = 7.4 Hz, 2H), 1.38 (s, 3H), 1.09 (t, J = 7.5 Hz, 3H). 13C NMR
(400 MHz, CDCl3): δ 172.4 (s), 140.2 (s), 139.0 (s), 136.0 (s), 135.7
(s), 133.2 (s), 132.7 (s), 128.1 (s), 127.9 (s), 127.8 (s), 127.8 (s),
126.8 (s), 126.5 (s), 126.2 (s), 123.2 (s), 22.8 (s), 16.7 (s), 13.5 (s),
13.4 (s). UV−vis (CHCl3): λ(nm) = 218 (ε = 42500 M−1 cm−1), 280
(ε = 16500 M−1 cm−1). EM-ES(+): (C19H19NO + H) calcd 278.1545,
found 278.1541. Observations: yellow solid.
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(E)-3-Ethyl-4-methyl-5-(1-(pyridin-2-yl)ethylidene)-1H-pyrrol-
2(5H)-one (E-2c). Yield = 52% (0.1186 g, mp 117−118 °C). 1H NMR
(400 MHz, CDCl3): δ 8.64 (d, J = 4.8 Hz, 1H), 7.78 (s, 1H), 7.70 (t, J
= 7.7 Hz, 1H), 7.30−7.27 (m, 1H), 7.25 (dd, J = 4.5, 3.1 Hz, 1H),
2.34−2.24 (m, 2H), 2.24 (s, 3H), 1.36 (s, 3H), 1.03 (t, J = 7.5 Hz,
3H). 13C NMR (100 MHz, CDCl3): δ 171.9 (s), 159.5 (s), 149.6 (s),
139.8 (s), 136.5 (s), 136.4 (s), 136.3 (s), 124.4 (s), 122.5 (s), 120.7
(s), 20.8 (s), 16.7 (s), 13.2 (s), 12.9 (s). UV−vis (CHCl3): λ(nm) =
292 (ε = 15000 M−1 cm−1). EM-ES(+): (C14H16N2O + H) calcd
229.1341, found 229.1345. Observations: yellow solid.
(Z)-3-Ethyl-4-methyl-5-(1-(pyridin-2-yl)ethylidene)-1H-pyrrol-
2(5H)-one (Z-2c). Yield = 18% (0.0411 g). 1H NMR (400 MHz,
CDCl3): δ 11.50 (d, J = 47.6 Hz, 1H), 8.58 (d, J = 4.7 Hz, 1H), 7.72
(t, J = 10.7, 5.3 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.14 (dd, J = 7.4, 4.9
Hz, 1H), 2.45−2.35 (m, 5H), 2.32 (s, 3H), 1.10 (t, J = 7.6 Hz, 3H).
13C NMR (400 MHz, CDCl3): δ 171.4 (s), 159.2 (s), 147.9 (s), 140.3
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(s), 139.4 (s), 137.1 (s), 136.9 (s), 122.0 (s), 121.2 (s), 113.2 (s), 16.7
(s), 16.3 (s), 15.6 (s), 13.6 (s). UV−vis (CHCl3): λ(nm) = 224 (ε =
11000 M−1 cm−1), 344 (ε = 34500 M−1 cm−1). EM-ES(+):
(C14H16N2O + H): calcd 229.1341, found 229.1345. Observations:
yellow solid.
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̈
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.6b00892.
■
(21) Quail, P. H. Annu. Rev. Genet. 1991, 25, 389.
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S
UV−vis spectra, X-ray data, DOSY experiments, hydro-
gen-bond strength evaluation, irradiation time vs Z/E
ratio plots, NMR spectra, and computational details
(PDF)
X-ray data for 1d (CIF)
X-ray data for 1f (CIF)
X-ray data for 2c (CIF)
(28) Mreihil, K.; McDonagh, A. F.; Nakstad, B.; Hansen, T. W. R.
Pediatr. Res. 2010, 67, 656.
AUTHOR INFORMATION
Corresponding Author
*E-mail: diego.sampedro@unirioja.es.
Notes
■
(29) Ennever, J. F. Photochem. Photobiol. 1988, 47, 871.
(30) Vreman, H. J.; Wong, R. J.; Stevenson, D. K. Semin Perinatol
2004, 28, 326.
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208, 145.
The authors declare no competing financial interest.
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1982, 35, 649.
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A.; Angeli, C.; Olivucci, M. J. Chem. Theory Comput. 2013, 9, 4495.
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Chem. A 2013, 117, 2790.
(35) Chuang, W.-T.; Hsieh, C.-C.; Lai, C.-H.; Lai, C.-H.; Shih, C.-W.;
Chen, K.-Y.; Hung, W.-Y.; Hsu, Y.-H.; Chou, P.-T. J. Org. Chem. 2011,
76, 8189.
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Phys. 2010, 12, 11051.
ACKNOWLEDGMENTS
This research was supported by the Spanish Ministerio de
■
Ciencia e Innovacion
Desarrollo Regional (FEDER) (CTQ2014-59650-P and
CTQ2012-36966) and Project CCG2013/EXP-089 for the
́
(MICINN)/Fondos Europeos para el
́
University of Alcala (UAH). C.G.-I. thanks the Spanish MEC
for her grant. Financial support by the Deutsche Forschungs-
gemeinschaft (GRK 2039) and KIT is gratefully acknowledged.
́
We thank Fernando Rodriguez Royo for his help in resolving
the X-ray structures.
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