Structural studies on the stereoisomerism of a natural dye miraxanthin I

Article abstract of DOI:10.1039/c9nj04215f

The chemical structure of a yellow dye present in Mirabilis jalapa L., miraxanthin I, was characterized by NMR spectroscopy. The extract of M. jalapa was analysed by a high-performance liquid chromatographic system (LC-DAD-FL-ESI-MS/MS) and the presence of miraxanthin I among other betaxanthins was confirmed. This dye was synthesized from previously generated methionine-betaxanthin by oxidation with 10% H₂O₂. The E/Z stereoisomers of miraxanthin I were found by NMR analysis to occur in a 50?:?33?:?10?:?7 ratio in aqueous solution (7E,9E?:?7Z,9E?:?7E,9Z?:?7Z,9Z configurations, respectively). Comparison of NMR data with chemical shifts obtained from quantum chemical calculations strongly suggests the presence of intramolecular hydrogen bonds which may favour a more rigid structure of the dye. This explains the highest fluorescence quantum yield among betaxanthins. The observed changes in the ¹H NMR spectra during the measurements indicate on miraxanthin I hydrolysis for which a mechanism is proposed. The first step during the hydrolysis process is protonation of the nitrogen atom within the central bridge bond system which is observed in the ZE stereoisomer by NMR.

Full text of DOI:10.1039/c9nj04215f

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Structural studies on the stereoisomerism of a
natural dye miraxanthin I
Cite this: New J. Chem., 2019,
43, 18165
b
c
Stanisław Nizinski,a Łukasz Popenda,
Agnieszka Kumorkiewicz,
Michał F. Rode,
´
d
e
f
Zbigniew Fojud,
Ewelina Paluch-Lubawa,
Sławomir Wybraniec *d and Gotard Burdzinski
*
a
´
The chemical structure of a yellow dye present in Mirabilis jalapa L., miraxanthin I, was characterized by
NMR spectroscopy. The extract of M. jalapa was analysed by a high-performance liquid chromatographic
system (LC-DAD-FL-ESI-MS/MS) and the presence of miraxanthin
I among other betaxanthins was
confirmed. This dye was synthesized from previously generated methionine-betaxanthin by oxidation with
10% H2O2. The E/Z stereoisomers of miraxanthin I were found by NMR analysis to occur in a 50 : 33 : 10 : 7
ratio in aqueous solution (7E,9E:7Z,9E:7E,9Z:7Z,9Z configurations, respectively). Comparison of NMR data
with chemical shifts obtained from quantum chemical calculations strongly suggests the presence of
intramolecular hydrogen bonds which may favour a more rigid structure of the dye. This explains the
highest fluorescence quantum yield among betaxanthins. The observed changes in the 1H NMR spectra
during the measurements indicate on miraxanthin I hydrolysis for which a mechanism is proposed. The first
step during the hydrolysis process is protonation of the nitrogen atom within the central bridge bond system
which is observed in the ZE stereoisomer by NMR.
Received 14th August 2019,
Accepted 25th October 2019
DOI: 10.1039/c9nj04215f
rsc.li/njc
industry as natural colorants7,8 or in DSSC (Dye Sensitized Solar
Cells) as photosensitizers.9,10 The structural properties of only a
Introduction
Betaxanthins are natural plant pigments which occur in most few betaxanthins have been studied so far. The most detailed
families of the Caryophyllales order. They give a yellow color to study in the past concerned indicaxanthin and miraxanthin V.11
inflorescences which helps to attract seed dispersers and Our recent efforts aimed at describing the photophysical proper-
pollinators.1 Miraxanthin I (MIR-I) is one of the betaxanthins ties of betaxanthins12–14 since their presence in plants may protect
found in inflorescences of Mirabilis jalapa L. (Fig. 1).2,3 The the photosynthetic apparatus against excess solar light intensity
flowers open in the late afternoon. Betaxanthins are formed by by forming a sun-screen filter. MIR-I is distinguished by
condensation of betalamic acid with an amine or amino acid the highest fluorescence quantum yield among the studied
which in the case of MIR-I is methionine sulfoxide.4 In betaxanthins.3 The unique photophysics of MIR-I is likely
solution, betaxanthins undergo degradation which can be influenced by dye structural factors. The basic structure of
slowed down by decreased temperature and pH in the range MIR-I is known (Fig. 1),2 nevertheless, the stereoisomeric
of 4–7.5,6 These dyes can be potentially applied in the food equilibrium has never been elucidated up to now. This paper
attempts to provide structural information on various MIR-I
1
forms populated in solution on the basis of H and 13C NMR
a Quantum Electronics Laboratory, Faculty of Physics, Adam Mickiewicz University
´
in Poznan, Uniwersytetu Poznanskiego 2, 61-614 Poznan, Poland.
E-mail: gotardb@amu.edu.pl
b NanoBioMedical Centre, Adam Mickiewicz University in Poznan,
Wszechnicy Piastowskiej 3, 61-614 Poznan, Poland
c
´
Institute of Physics, Polish Academy of Sciences, Aleja Lotnikow 32/46,
02-668 Warsaw, Poland
d Department of Analytical Chemistry, Institute C-1, Faculty of Chemical
Engineering and Technology, Cracow University of Technology, Warszawska 24,
31-155 Cracow, Poland. E-mail: swybran@chemia.pk.edu.pl
e Department of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz
´
University in Poznan, Uniwersytetu Poznanskiego 2, 61-614 Poznan, Poland
f Department of Plant Physiology, Institute of Experimental Biology,
Fig. 1 Yellow Mirabilis jalapa L. inflorescences, containing miraxanthin I
Faculty of Biology, Adam Mickiewicz University in Poznan,
´
and its structural formula.
Uniwersytetu Poznanskiego 6, Poznan, Poland
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investigations and theoretical calculations. NMR studies also methionine-betaxanthin reaction solution followed by rinsing
allowed us to describe the dynamics of the MIR-I hydrolysis with water and elution with 5% aqueous formic acid in 50%
process leading to methionine sulfoxide and betalamic acid acetonitrile. The eluate was pooled and pre-concentrated under
formation.
reduced pressure at 25 1C. Further cleanup of methionine-
betaxanthin was performed by semipreparative chromatography
on an HPLC system with LC-20AP pumps, a UV-vis SPD-20AV
detector and LabSolutions 5.51 operating software (Shimadzu
Corp., Kyoto, Japan). The semipreparative column used was a
250 mm ꢀ 30 mm i.d., 10 mm, Luna C18(2), with a 20 ꢀ 25 mm
i.d. guard column of the same material (Phenomenex). The
mobile phases were composed of 1% aqueous formic acid
(solvent A), and acetonitrile (solvent B). The gradient profile
was (t [min], % B), (0, 10), (20, 15), (30, 20), (40, 24). The
Experimental section
Semi-synthesis of methionine betaxanthin
As a natural substrate for semi-synthesis of MIR-I, indicaxanthin
(IND) from the orange–yellow fruits of Opuntia ficus-indica L. Mill.
was initially separated according to previously described reports.10,12
Briefly, after filtering, the juice of 1000 g O. ficus indica fruits was
chromatographically purified by open column chromatography on a
C18 sorbent (Merck, Darmstadt, Germany). The eluates were pooled
and preconcentrated under reduced pressure at 25 1C. The concen-
trated extract was subjected to semipreparative flash chromato-
graphy on a C18 flash column (Interchim, France) for separation
of IND. Further purification of the pigment was performed on
an HPLC semipreparative column Luna C18(2) (Phenomenex,
Torrance, CA, USA). The eluates were pooled and preconcentrated
under reduced pressure at 25 1C and finally freeze-dried to obtain
pure IND.
During the semi-synthesis (Scheme 1), methionine was
added in a 25-fold molar excess to an alkaline solution of
betalamic acid obtained directly after 30 mg IND hydrolysis at
pH 11.4 through the addition of 25% NH4OH. 20 min after
adding methionine, the solution was acidified to pH 7 by formic
acid and stirred for another 15 min. All the reactions were
performed at low temperature (10 1C) under an inert atmosphere
of argon.
injection volume was 25 mL, and the flow rate was 50 mL minꢁ1
.
The column was thermostated at 25 1C, and the detection was
performed at 538 and 480 nm. Separated fractions were pooled
and pre-concentrated under reduced pressure at 25 1C and finally
freeze-dried before further oxidizing the isolated pigment.
Purified methionine-betaxanthin (15 mg) was oxidized by
10% H2O2 leading to the formation of MIR-I (Scheme 1). The
reaction was carried out for 15 min and the obtained product
was immediately subjected to semipreparative chromatography
on the Shimadzu HPLC system under the following gradient
system consisting of 0.05% aqueous formic acid (solvent A) and
acetone (solvent B): (t [min], % B), (0, 1), (20, 2), (30, 4). The
injection volume was 25 mL and the flow rate was 50 mL minꢁ1
.
The column was thermostated at 25 1C and the detection was
performed at 440 and 480 nm. The fractions were pooled and
preconcentrated under reduced pressure at 25 1C and finally
freeze-dried to obtain pure MIR-I (9 mg).
After the semi-synthesis, the obtained methionine-betaxanthin
was chromatographically purified by ion-exchange chromato-
graphy on a cartridge filled with a strong anion exchanger
(Sepra ZT-SAX Phenomenex, Torrance, CA). The bed was pre-
conditioned with water before the application of the obtained
Plant material
M. jalapa germinated seeds were planted in 3 L pots (two plants
per pot) filled with substrate for vegetables (1.0 kg mꢁ3 NPK
14 : 16 : 18, neutralized to pH 5.5–6.0). The culture was carried
out in phytotron under controlled conditions (day/night 16/8 h,
23 1C, light intensity 150 mmol mꢁ2 ꢁ1, 60% humidity). Plants
s
were fertilized once a month using Hougland medium. The plant
material was collected immediately after complete opening of
flowers. Immediately after picking, the flowers from M. jalapa
were ground in ethanol (150–200 mg tissue for 0.5 mL ethanol).
Chromatographic analysis by LC-DAD-FL-ESI-MS/MS technique
For chromatographic and mass spectrometric analyses, an
LCMS-8030 mass spectrometric system (Shimadzu) coupled to
LC-20ADXR HPLC pumps, an injector model SIL-20ACXR,
a PDA detector (photo diode array) model SPD-M20A, and an
RF-20Axs fluorescence detector, all controlled with LabSolutions
software, version 5.60 SP1 (Shimadzu), was used. The samples
were eluted through a 150 mm ꢀ 4.6 mm i.d., 5.0 mm, Kinetex C18
chromatographic column preceded by a guard column of the
same material (Phenomenex). The injection volume was 20 mL,
and the flow rate was 0.5 mL minꢁ1. The column was thermo-
stated at 40 1C. The separation of the analytes was performed
with a binary gradient elution. The mobile phases were
composed of 2% aqueous formic acid (solvent A) and pure
Scheme 1 General reaction scheme for semi-synthesis of miraxanthin I.
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methanol (solvent B). The gradient profile (system 1) was applying low-power presaturation pulses during the relaxation
(t [min], % B), (0, 5), (12, 70), (15, 80), and (19, 80). For analysis delay or via the excitation sculpting technique. The 1H and 13
C
of the extract of yellow M. jalapa inflorescences, the gradient chemical shifts were determined relative to TMS (0.00 ppm for
profile (system 2) was (t [min], % B), (0, 2), (20, 13), (35, 25), 1H, 0.0 ppm for 13C) as an internal standard.
and (40, 80). The full range PDA signal was recorded, and
chromatograms at 538, 505, 490, and 440 nm were individually
displayed. The fluorescence chromatograms of M. jalapa
Results and discussion
NMR structural investigations of miraxanthin I
extract were recorded at lex 465 nm and lem 508 nm.3 Positive
ion electrospray mass spectra were recorded on the LC-MS
system, which was controlled with LabSolutions software. The For structural investigations of MIR-I, 1H and 13C NMR mea-
ionization electrospray source operated in positive mode at an surements were performed either in 90% H2O/10% D2O or in
electrospray voltage of 4.5 kV and capillary temperature at pure D2O solutions. Due to the low chemical stability of MIR-I
250 1C, using N2 as a gas for the spray, recording total ion compared to other betaxanthins, low temperature conditions
chromatograms, mass spectra, and ion chromatograms in were used (T = 7 1C), which facilitated time-consuming
selected ion monitoring mode (SIM) as well as the fragmentation experiments.
spectra. Argon was used as the collision gas for the collision-
The 1H and 13C NMR resonance assignments (Table 1) were
induced dissociation (CID) experiments. The relative collision established based on 1D 1H and 2D 1H–1H gCOSY, 1H–1H
energies for MS/MS analyses were set at ꢁ35 V.
TOCSY, 1H–1H NOESY, 1H–13C gHSQC, and 1H–13C gHMBC
experiments.
NMR measurements
Due to a variety of possible low-energy stable forms in which
The NMR spectra were recorded on an Agilent DD2 800 MHz the dye can co-exist, we distinguish them with respect to their
spectrometer, equipped with a 5 mm 1H{13C/15N} probe head and structural features, such as rotations about double or single
operating at resonance frequencies of 799.903 and 201.146 MHz bonds resulting in the ground-state energy barriers separating
for 1H and 13C, respectively. All spectra were measured at a these forms.
temperature of T = 7 1C unless otherwise stated. The measure-
Thus, the E/Z stereoisomers (7E,9E, 7E,9Z, 7Z,9E and 7Z,9Z,
ments were performed in 99.9% D2O at an approximate sample abbreviated as EE, EZ, ZE and ZZ, respectively, for the sake of
concentration of 0.01 M, or in D2O/H2O (10/90, v/v) at a sample simplicity) distinguish structures with respect to the rotations
concentration of 0.04 M. The 1D (1H, 13C) and 2D (1H–1H gCOSY, about the two double bonds present in the CQC–CQN chain.
1H–1H TOCSY, 1H–1H NOESY, multiplicity-edited 1H–13C
One specific stereoisomer may have different forms with
gHSQCAD and 1H–13C gHMBCAD) experiments were performed respect to the various intramolecular hydrogen bonds which
using standard Agilent pulse sequences and acquisition para- may be formed to stabilize its structure. The 1H spectra
meters. The 2D 1H–1H NOESY spectra were acquired with revealed sets of resonances characterizing four MIR-I stereo-
mixing times of 400, 600 and 800 ms. The 1H–1H TOCSY isomers: EE, ZE, EZ and ZZ (see Table 1 and Fig. 2).
experiments were collected using the MLEV-17 spin-lock
The individual proton spin systems H2–C(3) through
sequence for Hartman–Hahn transfer with a mixing time of H2–C(4) to H–C(5), H–C(8) to H–C(9), and H2–C(11) to H–C(12)
80 ms. Solvent suppression was accomplished either by were identified using the gCOSY and TOCSY techniques.
Table 1 1H and 13C NMR chemical shift assignments (ppm) for miraxanthin I (MIR-I) stereoisomers at T = 7 1C in D2O/H2O (10/90, v/v)
Miraxanthin I stereoisomer
EE (50%)
1H
ZE (33%)
1H
EZ (10%)
1H
ZZ (7%)
1H
Hydrolysis products
Atom no.
d
d
13C
d
d
13C
d
d
13C
d
d
13C
d
1H
d
13C
2
3
4a/b
2.74
3.01
2.33
2.49
4.50
—
37.5
48.9
26.4
2.74
2.98
2.34
2.48
4.67
—
9.51
8.12
6.08
—
3.39
3.14
4.56
—
6.24
—
37.5
49.3
25.5
2.74
3.01
2.33
2.49
4.56
—
37.5
48.9
26.7
2.74
2.98
2.34
2.48
4.57
—
37.5
49.3
—
2.77
3.04
2.37
37.3
49.0
24.6
5
6
7
8
63.2
174.6
—
161.4
108.8
164.3
27.4
57.3
174.5
—
156.5
105.1
166.2
27.3
63.4
—
—
162.6
107.9
165.0
33.9
58.1
—
—
157.4
104.0
—
4.07
—
—
9.58
5.85
—
3.35
3.16
4.41
—
53.6
173.4
—
189.9
120.3
157.9
—
—
—
—
8.22
6.17
—
3.37
3.10
4.52
—
6.18
—
—
8.56
5.88
—
3.16
3.09
4.45
—
6.55
—
—
8.46
5.75
—
3.16
3.08
4.45
—
6.58
—
—
9
10
11a/b
34.1
12
14
15
16
17
53.2
153.5
103.9
175.5
166.6
53.1
155.6
104.6
175.2
166.4
53.4
150.8
96.7
176.0
167.0
—
53.5
146.0
—
176.4
—
157.3
96.6
—
6.38
—
—
—
—
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Fig. 2 Chemical structures and atom numeration of miraxanthin I stereo-
isomers and hydrolysis products. Important NOE correlations are indicated
by arrows.
Connections between H–C(5) and H–C(8) via N(7), between
H3–C(2) and H2–C(3) via S(1), between H–C(9) and H–C(11), and
between H–C(9) and H–C(15) were elucidated on the basis of
1H–13C gHMBC experiments.
Analysis of the 1H–13C gHMBC spectrum yielded 2J(H,C) and
3J(H,C) scalar couplings, which made it possible to establish
correlations between H–C(5), H–C(12), H–C(15) and C(6), C(16),
C(17) atoms and to identify the COOH carbon resonances.
Progressive MIR-I degradation and the low concentration of
MIR-I minor stereoisomers (EZ and ZZ) precluded obtaining
directly time consuming 13C spectra, thus assignment of
13C resonances proceeded using a combination of gHSQC and
gHMBC experiments. Identification of 1H resonances was
supported by the presence of corresponding cross-peaks in
the 2D NOESY spectrum. Moreover, a Nuclear Overhauser
Effect (NOE) experiment was used to establish the stereo-
isomeric configuration of MIR-I, which was also supported
by quantum chemical calculations, described in detail later.
Observation of EXSY cross-peaks in the 2D NOESY spectrum
(Fig. 3A and B) indicated dynamic equilibrium of four E/Z
Fig. 3 The low-field regions (A and B) of the 2D 1H–1H NOESY spectrum
(800 ms mixing time) acquired for miraxanthin I in D2O/H2O (10/90, v/v) at
T = 7 1C are shown. The experiment shows cross-peaks due to exchange
between the four miraxanthin I stereoisomers in solution.
stereoisomers, which has been previously reported for populated one, whereas the presence of the cross-peak between
miraxanthin V and indicaxanthin.11
H–C(5) and H–C(9) confirmed the ZE configuration as the
The assignment and conformational analysis of the two second most stable stereoisomer (Fig. 2). Furthermore, detec-
minor (EZ and ZZ) stereoisomers are supported by much less tion of NOE cross-peaks between H–C(9) and H–C(11), H–C(5)
intense signals associated with a weak S/N ratio. The four E/Z and H–C(8) and H–C(8) and H–C(15) confirmed the presence of
stereoisomers of MIR-I occurred in a 50 : 33 : 10 : 7 ratio (EE :ZE : the EZ stereoisomer, while the appearance of a low intensity
EZ :ZZ, respectively), as calculated by integration of H–C(8) peaks signal between H–C(8) and H–C(15) indicated the ZZ stereo-
(because of their best chemical shift dispersion). Strong NOE isomer as the least abundant one (Fig. 2). A comparative
signals between H–C(8) and H–C(11) indicated a 9E configuration analysis of d(1H) of H2–C(11) additionally confirmed the 9E
of both major stereoisomers (EE and ZE), for which a correlation configuration of the two major stereoisomers and the 9Z
between H–C(9) and H–C(15) was also expected (Fig. 2). However, configuration of the minor ones. Analogously, the similarity
1
in these cases, almost identical chemical shifts for both of the H chemical shifts of H–C(15) leads to the same conclu-
resonances (Table 1) and/or overlapping with EXSY cross- sions. A comparison of d(13C) of C(11) and likewise C(15) was also
peaks prevented direct observation of the spin–spin couplings consistent with above observations. Based on the 13C chemical
through space. Additionally, the correlation between H–C(5) shifts obtained for C(5) similar deductions can be provided for the
and H–C(8) indicated the EE stereoisomer as the most EE and the EZ as well as for the ZE and the ZZ stereoisomers to
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Fig. 5 Miraxanthin I and betalamic acid concentration kinetics in D2O/
H2O (10/90, v/v); pH 3.7; T = 20 1C.
Fig. 4 Miraxanthin I and betalamic acid concentration kinetics in D2O;
pD 4.1; T = 7 1C.
justify the given 7-stereocenter configurations. Previously pub- starts (t = 0), and kM and kB are speed constants describing the
lished NMR data for other betaxanthins confirmed the consis- MIR-I and betalamic acid decomposition processes, respectively.
tency of the results obtained for the betalamic acid moiety.11,15,16 The solution of this equation under the circumstance [B] = 0 at
Moreover, the determined 3J(H,H) coupling constants between t = 0 consists of two exponential terms:
H–C(8) and H–C(9) were 12.0, 12.8, 11.9 and 12.6 Hz, for the EE,
À
Á
kM
Bt
Mt
½Mꢂ0 eꢁk ꢁ eꢁk
ZE, EZ and ZZ stereoisomers, respectively, and were in good
agreement with couplings obtained for another betaxanthin,
miraxanthin V (11.9, 12.6, 12.0 and 12.5 Hz), indicating an s-trans
conformation of the dienyl moieties.11
½Bꢂ ¼
kM ꢁ kB
Two different theoretical possibilities can be distinguished:
kM 4 kB or kB 4 kM. The second case can be applied for kinetics
interpretation, assuming that betalamic acid degrades faster
than MIR-I. In such conditions, the betalamic acid concen-
tration grows with a time constant associated with its degrada-
tion and later undergoes a decay with a time-constant equal to
the MIR-I population lifetime. In our experiment, the deter-
mined time constants are tM = kMꢁ1 = 132 h and tB = kBꢁ1 = 24 h
at low temperature conditions (T = 7 1C).
Thus, the MIR-I hydrolysis process is faster at 20 1C than at
7 1C. This trend is consistent with the known increase in the
rate of betaxanthin degradation in response to a temperature
rise.6 We propose the MIR-I degradation mechanism presented
in Scheme 2, based on a similar case recently reported for a
structurally similar dye – betanin.18 Upon dynamic equilibrium
between MIR-I stereoisomeric forms, the hydrolysis process
likely proceeds for the most reactive dye form. Protonation of
MIR-I at N(7) may facilitate the hydrolysis process. Indeed, we
found strong COSY correlations between the peak located at
9.51 ppm and signals at 8.12 and 4.67 ppm, which correspond
to H–C(8) and H–C(5) of the ZE stereoisomer, respectively. This
suggests the presence of a proton bound to N(7) in the ZE
stereoisomer. Integration of the 9.51 and 8.12 ppm peaks
suggests about a 5% contribution of a form possessing an
H-N(7) proton to the overall ZE stereoisomer population. The
presence of the H-N(7) proton in the ZE stereoisomer weakens
the N(7)QC(8) bond by about 0.03 Å, according to DFT calcula-
tions. In such conditions, the protonated MIR-I molecule might
Similarly to the betaxanthins analyzed by Stintzing et al.,11
we observed H/D exchange for H–C(9) and H–C(15) for MIR-I
1
in D2O solution which can be seen in Fig. 4 where H spectra
were collected as a function of time. Moreover, occurring of
a hydrolysis reaction caused MIR-I degradation, resulting in a
decrease of concentration and the appearance of degradation
products which were detected by the NMR technique. Repre-
sentative well-separated signals were chosen and integrated
to obtain kinetic profiles of this process. The two time
constants: t1 = 7.2 h and t2 = 43 h represent the deuteration
process of H–C(15) and H–C(9) protons, respectively. Analo-
gous data recorded in D2O/H2O (10/90, v/v) and at a higher
temperature of T = 20 1C (Fig. 5) show a single exponential
decay for H–C(9) and H–C(15), with a time constant tM = 22 h.
A faster H/D exchange process for H–C(15) was assumed based
on the similar case of betanidin.17 Time constant tM corre-
sponds to the MIR-I lifetime, which is longer (132 h) at low
temperature T = 7 1C. MIR-I undergoes the hydrolysis process
leading to a formation of betalamic acid and methionine
sulfoxide.
Betalamic acid is not a persistent product but undergoes
further reactions. An equation describing the evolution of the
betalamic acid population is the following:
d½Bꢂ
¼ kM½Mꢂ ꢁ kB½Bꢂ ¼ kM½Mꢂ0eꢁk ꢁ kB½Bꢂ
Mt
dt
where [B] is the betalamic acid concentration, [M] is the MIR-I be more prone to nucleophilic attack of water at electrophilic
concentration, [M]0 is the MIR-I concentration before hydrolysis C(8). The proposed mechanism of MIR-I hydrolysis as a result
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Fig. 6 The EE stereoisomeric forms of miraxanthin I; from the left side:
Open form, structure with a COOHꢃ ꢃ ꢃN hydrogen bond, and with a
SOꢃ ꢃ ꢃHOOC hydrogen bond, as well as the harpoon form with a hydrogen
bond between the MetSO and betalamic acid moiety.
for each MIR-I E/Z stereoisomer. Those are e.g. EES, EEN, EEo and
EEh, for the EE stereoisomer (Fig. 6). Those forms can be
distinguished by a lack or the presence of different intramolecular
hydrogen bonds that might be formed in the MIR-I molecule.
The least stable is the Open form (EEo, Fig. 6) in which structure
there is no intramolecular hydrogen bond present.
Any intramolecular hydrogen bond formed in the structure
of the molecule can give additional energetic stabilization in
comparison to the Open form. One possibility is a hydrogen
bond between nitrogen atom N7 in the central bridge and
the nearby C(5)-substituted carboxyl group (the EEN form,
Fig. 6). The same carboxyl group can form another hydrogen
bond with the oxygen atom present in the sulfoxide group
creating the hydrogen bonded eight-membered ring in the EES
form (Fig. 6).
Finally, the most compact form can be obtained when the
characteristic harpoon-like hydrogen bond between the part
derived from betalamic acid and the MetSO moiety is present
(EEh, Fig. 6). Indeed, this harpoon EEh form is stabilized
by two intramolecular hydrogen bonds – the second one is
the N ꢃ ꢃ ꢃHOOC bond, which is present also in the EEN form
(Fig. 6). The lowest energy conformer for all the proposed
forms was optimized for each E/Z stereoisomer. The obtained
Scheme 2 Proposed mechanism of miraxanthin I hydrolysis as a result of
its protonation at N(7) postulated for the ZE stereoisomer.
of its protonation at N(7) (the ZE stereoisomer) is presented in dipole moments and relative energies are summarized in
Scheme 2. The protonation leads to formation of the iminium Table 2.
cation, which undergoes a nucleophilic attack by water at
To obtain reliable information about the MIR-I populations
carbon C(8) followed by a transfer of the proton from attached of different stereoisomers in solution and strengthen the NMR
water to nitrogen N(7) and elimination of the amino acid with assignment, 1H NMR chemical shifts were calculated using the
final deprotonation of the formed betalamic acid molecule.
GIAO method (Gauge-Independent Atomic Orbital).27–29 The
lowest-energy conformer was chosen as the most representative
for each intramolecular-hydrogen bond form (o, N, S, h) of a
Quantum chemical calculations
Quantum chemical calculations using Density Functional given stereoisomer (EE, EZ, ZE, ZZ, see Table 2). Root-mean-
Theory (DFT) were performed to predict the most stable conformers square deviation (RMSD) values between the chemical shifts
for each of the four stereoisomers of MIR-I and compare them obtained theoretically (for the representative conformer in a
with those determined in NMR spectra. Different geometries given form of a given E/Z stereoisomer) and experimentally
were optimized using the DFT method with the TPSSh hybrid (for a given E/Z stereoisomer) were calculated. The Open form
functional19–21 and 6-311++G(d,p) basis set.22,23 To mimic water of the EE stereoisomer structure with two explicit water
solvent conditions, the polarizable continuum model24,25 was molecules added to the system was also optimized to provide
employed in those calculations as implemented in the Gaussian 09 certainty that the solvent is not effectively competing with
software package.26 Various different starting geometries were intramolecular hydrogen bond formation. Intermolecular
used to find the lowest energetic conformers for each stereo- hydrogen bonds were placed between the C(6)OOH group and
isomer. We found that there are at least four different types of the first H2O as well as between N(7) and the second H2O, to
conformers – which we name forms – that should be considered saturate spots prone to hydrogen bond creation.
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Table 2 Relative energies and dipole moments of miraxanthin I depending on the stereoisomeric configuration and type of hydrogen bond calculated
by TPSSh/6-311++G(d,p) with PCM = water for the energetically lowest geometries
Intramolecular hydrogen bond form of the E/Z stereoisomer
E/Z
SOꢃ ꢃ ꢃHOOC
Nꢃ ꢃ ꢃHOOC
Open
Closed (harpoon)
EE
E = 115 cmꢁ1
m(S0) = 12.1 D
EES
E = 409 cmꢁ1
m(S0) = 11.9 D
EEN
E = 2459 cmꢁ1
m(S0) = 8.2 D
EEo
E = 0 cmꢁ1
m(S0) = 4.6 D
EEh
ZE
EZ
ZZ
E = 1208 cmꢁ1
m(S0) = 7.9 D
ZES
E = 998 cmꢁ1
m(S0) = 12.8 D
ZEN
E = 3927 cmꢁ1
m(S0) = 4.8 D
ZEo
E = 290 cmꢁ1
m(S0) = 6.4 D
ZEh
E = 312 cmꢁ1
m(S0) = 8.8 D
EZS
E = 629 cmꢁ1
m(S0) = 12.3 D
EZN
E = 2660 cmꢁ1
m(S0) = 5 D
EZo
E = 722 cmꢁ1
m(S0) = 9.8 D
EZh
E = 1398 cmꢁ1
m(S0) = 10.4 D
ZZS
E = 1212 cmꢁ1
m(S0) = 12 D
ZZN
E = 4146 cmꢁ1
m(S0) = 8 D
ZZo
E = 63 cmꢁ1
m(S0) = 6.4 D
ZZh
Table 3 1H NMR signals (ppm) for the EE stereoisomer from the NMR experiment compared with the results of quantum chemical calculations using
GIAO, TPSSh/6-311++G(d,p), PCM = water
Atom
Experiment
SOꢃ ꢃ ꢃHOOC
COOHꢃ ꢃ ꢃN
Closed
Open
Open + explicit water
2
3
4a
4b
5
8
9
11a
11b
12
15
RMSD*
2.74
3.01
2.33
2.49
4.50
8.22
6.17
3.37
3.10
4.52
6.18
—
2.56
3.06
2.03
2.50
4.58
8.54
6.37
3.61
2.63
4.17
6.58
0.275
2.37
2.48
1.86
2.55
4.15
8.53
6.37
3.59
2.68
4.22
6.54
0.349
2.48
2.79
1.49
2.36
3.76
8.38
6.45
3.25
2.92
4.23
6.50
0.396
2.40
2.51
1.70
2.33
4.76
8.35
6.39
3.54
2.62
4.16
6.54
0.363
2.48
2.65
1.72
2.33
4.68
8.40
6.39
3.54
2.61
4.15
6.56
0.339
The results of the calculation are presented in Table 3, for
There is a possibility that both the forms can be simulta-
the EE stereoisomer, and in Table 4, for all four stereoisomers. neously populated. In the case of the ZZ stereoisomer, all RMSD
For every E/Z stereoisomer, we picked one or two best candi- values are small (under 0.3), which makes it harder to select the
dates (values for the given conformer marked by bold font in best theoretical representative. The reason may be inaccurate
Table 4) which have relatively small RMSD values. Note that in experimental data from experiment due to the low intensity
most cases, the RMSD values in Table 4 reflect the E/Z stereo- of signals. Nevertheless, the Open and COOHꢃ ꢃ ꢃN forms can
isomeric assignment deduced only from experimental NMR be excluded from consideration due to the high RMSD. It is also
data analysis (so the lowest RMSD values are lying close to worth noting that the tested EEo form with the explicit water
diagonal). This relationship is slightly violated in some cases molecules possesses also a high RMSD, therefore, we suppose
but we are inclined to trust the E/Z stereoisomeric assignment that intermolecular hydrogen bonds with the solvent are not
obtained from experimental data and use Table 4 only to favoured over the intramolecular SOꢃ ꢃ ꢃHOOC hydrogen bond.
resolve which form of each E/Z stereoisomer is the most likely We can conclude that only SOꢃ ꢃ ꢃHOOC and the harpoon
populated in solution and which one can be excluded from forms should be considered but we cannot unambiguously
consideration. resolve which one is more populated. Population of these
Therefore, we conclude that all assignments in Table 4 forms with intramolecular hydrogen bonds in solution,
which are not compatible with experimental assignment of especially the harpoon one, would lead to suppressed twisting
E/Z stereoisomers (marked in italics) can be reasonably motion about the central bridge bonds, which explains why the
excluded. In the case of the stereoisomers with the 7E configu- radiationless excited state relaxation pathway is decelerated for
3
ration (EE and EZ), the SOꢃ ꢃ ꢃHOOC form turned out to have MIR-I in comparison to other betaxanthins as confirmed
decidedly the lowest RMSD value which allows us to postulate herein by a substantially high fluorescence quantum yield
that this form is the most populated. In the case of the (FF = 0.0084 in H2O) in comparison to betaxanthins recently
stereoisomers with the 7Z configuration (ZE and ZZ), we found studied (FF = 0.003 for miraxanthin V, FF = 0.0053 for indicax-
that the harpoon form can compete with the SOꢃ ꢃ ꢃHOOC anthin and FF = 0.0073 for vulgaxanthin I in H2O).12–14
form and we were not able to distinguish which form is more This finding agrees with the properties of the structural analogue
probable.
betacyanins showing that the presence of intramolecular
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Table 4 Root-mean-square deviation (RMSD) values calculated between
experimental and theoretical chemical shifts (GIAO, TPSSh/6-311++G(d,p),
PCM = water) for different forms of the four miraxanthin I stereoisomers.
Our best matches for each stereoisomer are marked in bold. Values under
0.3 (best coincidence) are marked green, between 0.3 and 0.35 are
marked yellow, between 0.35 and 0.4 – orange, and over 0.4 – red.
Values incompatible with experimental assignment to the E/Z stereo-
isomers are marked in italics
Fig. 7 Chromatographic profiles (System 2) of Mirabilis jalapa L. inflores-
cence extract with detection by fluorescence emission at 508 nm (A),
visible light absorption at 465 nm (B) and mass spectrometry in selected
ion monitoring (SIM) mode (C) for protonated molecular ions at m/z 359
(for MIR I, 1), 347 (for MIR V, 3), 331 (for MIR III, 4) and 361 (for 3-methoxy-
tyramine-Bx, 5) in the LC-DAD-FL-ESI-MS/MS system.
these groups to reproduce chemical shifts correctly. In addition, it
would also require testing all the combinations of deprotonated
groups because the order of deprotonation is still unclear in the
case of betaxanthins. We plan to perform such calculations in the
hydrogen bonds extends the lifetime of the excited singlet future for some betaxanthins with a simpler structure such as
state in solution.30 Recent experimental and theoretical alanine-betaxanthin.32
studies reveal that the betaxanthin S1 state deactivation
Chromatographic analysis of betaxanthin extracts and
synthesized samples
mechanism involves torsional relaxation around the central
bonds (NQC and/or CQC).14,31 Herein, the presence of the
intramolecular hydrogen bond in the harpoon form of MIR-I The chromatograms (Fig. 7) of yellow Mirabilis jalapa L. inflores-
can hinder the specific bond rotations involved in the cence extract monitored by three methods, fluorescence emission,
non-radiative relaxation of the excited state. The existence of visible light absorption and mass spectrometry, clearly show a
so many different conformers of MIR-I that can be possibly strong rise of the fluorescence emission signal for MIR-I in
populated in solution generates some confusion. Nevertheless, comparison to the other methods in good agreement with
we can conclude that population of the ‘‘harpoon’’ or Gandia-Herrero et al.3
SOꢃ ꢃ ꢃHOOC forms is much more probable than the rest of
The other dyes detected (Table 5) in the extract are
the proposed forms under consideration. The forms for which dopamine-Bx (miraxanthin V),11 present at the highest concen-
twisting motion would be constrained in such a way have never tration, as well as tyramine-Bx (miraxanthin III) and 3-methoxy-
been shown in the case of other betaxanthins, which makes tyramine-Bx, but also unknown compounds absorbing light in
MIR-I unique among them.
the betaxanthin characteristic wavelength range. In order to
Better compatibility with theoretical results could be probably perform detailed spectroscopic studies MIR-I was synthesized
achieved by correctly deprotonating some carboxylic groups but it from methionine based betaxanthin (Fig. 8, Table 5) by its
would demand taking into account explicit solvent molecules near oxidation with 10% H2O2 and subjected to purification.
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Table 5 Chromatographic (System 2), spectrophotometric and mass spectrometric data of the analyzed betaxanthins from M. jalapa L. extract as well as
synthesized by amino acid exchange
No.
Compound
Retention time [min]
lmax [nm]
m/z [M + H]+
m/z from MS/MS of [M + H]+
1
2
3
4
5
Methionine sulphoxide-Bx (Miraxanthin I)
Methionine-Bx
Dopamine-Bx (Miraxanthin V)
Tyramine-Bx (Miraxanthin III)
3-Methoxy-tyramine-Bx
6.7
467
469
459
461
463
359
343
347
331
361
315; 271
299; 255
303; 259
287; 243
317; 273
11.6a
18.3/15.2a
24.2
24.9
a
Retention times obtained in chromatographic System 1.
The presented herein NMR analysis of the dye degradation
process due to hydrolysis can be used for other dyes belonging
to the betaxanthin family.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This research was supported in part by PL-Grid Infrastructure.
This work was performed with financial support of the Polish
National Science Centre (NCN), project DEC-2013/09/B/ST4/
00273.
Fig. 8 Chromatograms (System 1) of selected ions in the LC-ESI-MS/MS
system, monitored at m/z 359 (for MIR I, 1) and m/z 343 (for methionine-
Bx, 2), obtained for a mixture of 1 and 2 after 10 min of MIR I synthesis by
oxidation of 2 with 10% H2O2.
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