The thermal and enzymatic taxifolin-alphitonin rearrangement

Article abstract of DOI:10.1021/np200639s

This report describes a detailed investigation of the thermal and enzymatic conversion of taxifolin to alphitonin. Chromatographic separation of the four dihydroquercetin stereoisomers 1-4 in combination with circular dichroism spectroscopy permitted elucidation of the kinetics of this rearrangement and characterization of the different reaction pathways involved. Our findings are corroborated by quantum chemistry calculations that reveal a unique cascade of tautomerization processes leading from taxifolin to alphitonin and also explain the racemization of alphitonin at room temperature. Furthermore, the substrate specificity toward (+)-taxifolin of an enzyme from Eubacterium ramulus catalyzing this intriguing rearrangement is demonstrated.

Full text of DOI:10.1021/np200639s

Article
pubs.acs.org/jnp
The Thermal and Enzymatic Taxifolin−Alphitonin Rearrangement
Paul W. Elsinghorst,*,†,§ Taner Cavlar,† Anna Muller,† Annett Braune,‡ Michael Blaut,‡
̈
and Michael Gutschow†
̈
†Pharmaceutical Chemistry I, Pharmaceutical Institute, University of Bonn, 53121 Bonn, Germany
§Central Institute of the Bundeswehr Medical Service Munich, 85748 Garching-Hochbruck, Germany
‡Department of Gastrointestinal Microbiology, German Institute of Human Nutrition Potsdam-Rehbruecke, 14558 Nuthetal,
Germany
̈
ABSTRACT: This report describes a detailed investigation of
the thermal and enzymatic conversion of taxifolin to
alphitonin. Chromatographic separation of the four dihydro-
quercetin stereoisomers 1−4 in combination with circular
dichroism spectroscopy permitted elucidation of the kinetics of
this rearrangement and characterization of the different
reaction pathways involved. Our findings are corroborated
by quantum chemistry calculations that reveal a unique cascade
of tautomerization processes leading from taxifolin to
alphitonin and also explain the racemization of alphitonin at room temperature. Furthermore, the substrate specificity toward
(+)-taxifolin of an enzyme from Eubacterium ramulus catalyzing this intriguing rearrangement is demonstrated.
olyphenols exhibit several positive effects on human health,
e.g., antioxidant, antimicrobial, antiviral, and antitumor
e.g., alphitonin, that were not present in the original material.
However, the occurrence of alphitonin in the heartwood of A.
excelsa was affirmed, as it was also detected after cold
extraction.5 Recently, investigations of the intestinal metabo-
lism of quercetin revealed that the human gut bacterium
Eubacterium ramulus transforms quercetin to taxifolin and
subsequently to alphitonin.22 The same metabolites were
observed with the human colonic microbiota.23 Since quercetin,
mainly as glycosides, is abundant in food and beverages,
alphitonin deserves particular attention with respect to
formation, further transformation, and biological activity. The
proposed mechanism of the thermal taxifolin−aphitonin
rearrangement is outlined in Scheme 1, with special attention
to the possible dihydroquercetin stereoisomers.16,18,19,21,22
The determination of the absolute configuration of the four
dihydroquercetin stereoisomers and of some of its glycosides
has been the subject of several attempts. The corresponding
assignments were based on experimental results from optical
rotary dispersion and circular dichroism (CD) measurements,
and specifications of the absolute configuration rely either on a
comparison with the data of (+)-catechin or on theoretical
considerations.16,24−33 The CD spectra reported by Nonaka et
al.28 were used as the reference for the computational studies of
this report.
P
activities. A variety of health-promoting products containing
polyphenols are now available in the market as dietary
supplements.1,2 These polyphenols either are absorbed in the
human intestine in their original form or may be subject to
microbial metabolism.3,4 The two polyphenols in focus of this
report are taxifolin and alphitonin, with the former representing
a 4-chromanone, i.e., a dihydroflavonol, and the latter a 3(2H)-
benzofuranone, i.e., an auronol. While dihydroflavonols are
found in many plant families, auronols are much more limited
in their abundance and are especially present in Rhamnaceae.
Both classes of compounds usually occur as glycosides, and
notable auronol representatives are alphitonin, maesopsin,
amaronols A and B, zeyherin, marsuposide, and hovetricho-
side.5−13 Alphitonin was isolated first in 1922 from the wood of
Alphitonia excelsa, a common Australian tree.5,14 Taxifolin, also
referred to as dihydroquercetin, was named after the Douglas
fir, Pseudotsuga taxifolia, from which it was isolated first.15 It has
been recognized as a constituent of heartwood and needles in
Pinaceae, where it contributes to resistance against fungi and
termites.15−18 Both compounds are tightly linked to each other
by (i) a thermal and (ii) an enzyme-catalyzed rearrangement
reaction.
The observation that auronols can be obtained by treatment
of dihydroflavonols with alkali19,20 was further generalized in
1995 by Kiehlmann et al., who obtained alphitonin by heating a
neutral, aqueous solution of taxifolin for several hours, although
an oxidative side reaction occurred producing quercetin.19−21
Ohmura et al. corroborated this observation by detecting
alphitonin after steaming of taxifolin-containing wood.18 Hence,
steaming of wood, a common procedure in timber production,
or hot extraction of wood might lead to formation of auronols,
To investigate the thermal and enzymatic taxifolin−
alphitonin rearrangement, we established a chromatographic
system to separate all four dihydroquercetin stereoisomers.
Quantum chemical calculations were performed to assign their
conformations and to analyze the racemization process and the
Received: July 31, 2011
Published: October 12, 2011
© 2011 American Chemical Society and
American Society of Pharmacognosy and
American Society of Pharmacognosy
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Scheme 1. Proposed Route of the Rearrangement of Dihydroquercetins (1−4) to Alphitonin (8) via the Quinone Methides 5,
the Chalcone 6, and the Diketone 7
limiting energy barriers. The enzymatic conversion of taxifolin
to alphitonin was characterized with respect to its substrate
specificity and kinetic profile.
for the prediction of the CD spectra of (−)-(2R,3S)-epitaxifolin
(4) and (−)-(2S,3S)-taxifolin (2). Both diastereomers were
subjected to potential energy surface (PES) scans at the
B3LYP/TZVP computational level applying the COSMO
model in methanol and varying the two conformational
processes, i.e., a C-ring flip leading to equatorial/axial inversion
(φ1 = C6−C7−C8−O9) and a full rotation of the B−C-ring
bond (φ2 = O9−C8−C10−C11) (Figure 2A,B). From these
PES scans, local minima that are significantly populated at
ambient temperature (energy cutoff 3 kcal/mol) were identified
according to Boltzmann statistics and further optimized (Table
2). Finally, CD spectra of these minima were retrieved by a
TDDFT/B3LYP/TZVP approach and weighted according to
their individual Boltzmann population derived from energies at
the RI-SCS-MP2/TZVP computational level (Figure 2D). The
CD spectra obtained by these quantum chemical calculations
were in good agreement with the reported literature spectra.
Thus, our preliminary assumption of a fast keto−enol
tautomerism within the C ring was proven to be wrong. The
configuration of the temporarily enriched epitaxifolin is 2S,3R
(compound 3), and it is formed by C-2 epimerization.21,35
Whereas the separation of the four dihydroquercetin
stereoisomers was successfully achieved, several attempts to
separate the two putative alphitonin enantiomers by chiral
HPLC analysis failed, even with applying different HPLC
gradients and modifying the temperature. We also performed
chiral electrokinetic chromatography, where the alphitonin peak
again remained unresolved (Figure 1C). To explain this
observation, a fast on-column racemization was considered
and investigated in detail. Alphitonin racemization was assumed
to proceed by ring-opening to the diketone 7, being in a keto−
enol equilibrium with the α-hydroxychalcone 6 (Scheme 1). To
prove this assumption, an NMR experiment involving
deuterium incorporation at the benzylic position of alphitonin
was envisaged, as it had been successfully applied in an
equilibrium study of a related benzofuranone.6 Thus, a sample
of alphitonin obtained by thermal conversion was dissolved in
RESULTS AND DISCUSSION
■
Our initial attempts were directed toward the design of a
suitable reaction system to investigate the thermal taxifolin−
alphitonin rearrangement above 100 °C over a 14-day period.
To minimize oxidative quercetin formation, a reaction setup
was devised as follows. Taxifolin was placed in an amber glass
vial under argon, and oxygen-free water was added. The vial
was submersed in a solution of gallic acid as an external
protection from oxidation. The mixture was heated at 115 °C in
an oil bath, and aliquots were removed and subjected to
reversed-phase HPLC analysis.
Baseline separation of the four dihydroquercetin stereo-
isomers and alphitonin was achieved with an established
chromatographic system. Using cellulose tris(4-methylben-
zoate) as the chiral stationary phase, all analytes were separated
with sufficient resolution and plate numbers (Figure 1A, Table
1). In addition, CD spectra were recorded for each peak and
the absolute configuration of the four dihydroquercetin
stereoisomers was assigned on the basis of the work of Nonaka
et al.28 However, during thermal conversion of (+)-(2R,3R)-
taxifolin (1), the temporary enrichment of one epitaxifolin
enantiomer was observed. While the formation of one
epitaxifolin enantiomer 4 would result only from a keto−enol
tautomerization within the C ring,32 formation of the other
epitaxifolin enantiomer 3 would require ring-opening to a
quinone methide and recyclization (Scheme 1). Therefore, we
expected the temporarily accumulating epitaxifolin enantiomer
to be (−)-(2R,3S)-epitaxifolin (4). However, the observed CD
Cotton effect at 298 nm was positive, whereas literature
assignments require a negative Cotton effect for (−)-(2R,3S)-
epitaxifolin (4) at this wavelength. At this point, we were
prompted to perform quantum chemical calculations using the
ORCA computational package considering further literature34
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Figure 1. (A) Chiral separation of the dihydroquercetin stereoisomers
and alphitonin by RP-HPLC. The chromatogram was recorded in the
course of the thermal conversion of racemic (±)-taxifolin to
alphitonin. (B) Online CD detection coupled to chiral RP-HPLC.
The chromatogram was recorded in the course of the thermal
conversion of enantiopure (+)-taxifolin to alphitonin. (C) Chiral
electrokinetic chromatography of the dihydroquercetin stereoisomers
and alphitonin. The chromatogram was recorded in the course of the
thermal conversion of racemic (±)-taxifolin to alphitonin.
Figure 2. Potential energy surface (PES) scans of (−)-taxifolin (A)
and (−)-epitaxifolin (B) to simulate (i) a C-ring flip leading to
equatorial/axial inversion (φ 1 = C6−C7−C8−O9) and (ii) a full
rotation of the B−C-ring connecting bond (φ 2 = O9−C8−C10−
C11). Local minima are indicated in the contour plots (see also Table
2). (C) Selected stereoconformers of (−)-epitaxifolin with denoted
dihedral angles φ1 and φ2. (D) The predicted CD spectra (shown
here for (−)-taxifolin) are in good agreement with experimental
observations and corroborate the reported stereochemical assign-
ments.28
Table 1. Chromatographic Parameters of the Four
Dihydroquercetin Stereoisomers
resolution to
following peak
(min)
retention
time (min)
theoretical
plates
compound
(+)-epitaxifolin (3)
(−)-taxifolin (2)
(+)-taxifolin (1)
27.9
29.9
31.6
33.4
20300
25600
31100
33800
2.6
2.4
2.4
(−)-epitaxifolin (4)
protons (5.8−5.9 ppm), the latter being exchanged due to
keto−enol tautomerism (Figure 3A).36 Concerning the keto−
enol tautomerism of 6 and 7, this result did not support the
postulated 8 ⇌ 7 ⇌ 6 equilibrium (Scheme 1) and was also in
disagreement with literature reports of keto−enol tautomerism
in maesopsin.6,37
D2O, and the H/D exchange was followed by subsequent NMR
recordings. However, even after one week at room temperature,
no exchange of the benzylic protons was observed, in contrast
to the complete exchange of all phenolic and even of all A-ring
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Table 2. Local Minima from Conformational Analysis of
a
(−)-Taxifolin (2) and (−)-Epitaxifolin (4)
conformer
type
φ1 (deg)
φ2 (deg)
Ni (%)
2a
2b
2c
2d
2e
4a
4b
4c
4d
equatorial
equatorial
axial
57.0
57.1
236.5
58.8
41.31
56.68
0.74
−51.8
−48.8
−49.2
51.9
306.7
199.6
12.4
axial
0.63
axial
0.63
axial
282.0
75.1
18.52
32.61
27.32
21.54
axial
54.4
equatorial
equatorial
−53.7
−54.0
315.4
138.2
a
Figure 4. Quantum chemistry calculations for the interconversion of 2
to 8 reveal the ring-opening to the intermediate quinone methide 5 as
the energy barrier for the taxifolin−alphitonin rearrangement.
Data were obtained at the B3LYP/TZVP level of theory applying a
COSMO model in methanol. φ1 refers to the dihedral angle C6−C7−
C8−O9 (C-ring flip) and φ2 to O9−C8−C10−C11 (B−C-ring bond
rotation). Ni is the corresponding Boltzmann population used for
weighting of CD spectra.
Figure 5. Transition state 9 involving two water molecules was found
to facilitate racemization of alphitonin (8) via diketone 7.
chalcone 6. Using the Gaussian program suite38 at the B3LYP/
6-311G++(df,pd) computational level, the transition state 9
connecting 7 and 8 was investigated taking a possible proton
relay by one or two water molecules into consideration (Figure
5; Table 3).39 The transition state involving two water
molecules represents an energy barrier that can be readily
overcome at room temperature (2.34 kcal/mol), and thus,
alphitonin (8) may racemize at room temperature via the
intermediate diketone 7. This result is in accordance with the
observation that auronols are usually obtained as racemates but
can be resolved after derivatization of the hemiacetal hydroxy
group.40−42 However, as the existence of α-hydroxychalcone 6
is very short-lived, practically no deuterium is incorporated
during racemization in D2O.
Chiral chromatographic analysis of the in-process control
samples taken during thermal and enzymatic conversion
revealed a first-order mechanism for the taxifolin−alphitonin
rearrangement (Figure 6A,B). A pronounced enantioselectivity
toward (+)-taxifolin was observed for the enzyme from E.
ramulus accompanied by a high turnover rate, which was 200-
fold accelerated at room temperature in the presence of 1.9 μg
protein/mL of the partially purified enzyme (k = 7.26 × 10−4
s−1, Figure 6B) when compared to thermal conversion at 115
°C (k = 3.60 × 10−6 s−1, Figure 6A). Due to the observed fast
racemization of alphitonin, no conclusions could be drawn on
stereoselective product formation. The further characterization
of the enzyme from E. ramulus will be the subject of future
work. Aurone derivatives exhibit a notable therapeutic potential,
e.g., as antiviral, immunosuppressive, and chemopreventive
agents or as inhibitors of multidrug resistance.43−47 The
thermal and enzymatic conversion reported herein will be
useful to provide alphitonin for future biological evaluation.
Figure 3. (A) 1H NMR spectra of alphitonin immediately after
dissolving (top) and after one week of incubation (bottom) in D2O.
(B) MS fragmentation of alphitonin (see also Experimental Section).
To reconfirm the auronol structure of 8, MS analysis was
performed and corroborated its cyclic, and thus chiral,
benzofuranone structure (Figure 3B; see also Experimental
Section). Once again, we applied quantum chemical
calculations to gain further insight into the equilibrium
processes connecting the two alphitonin enantiomers to each
other and to the four dihydroquercetin stereoisomers.
Using the ORCA program suite, minimum energy con-
formers were optimized for 2, the assumed intermediates 5−7,
and 8 at the B3LYP/TZVP level of theory applying a COSMO
water model, followed by frequency calculations at 25 and 115
°C. The obtained Gibbs free energies confirmed the obvious
assumption that ring-opening of taxifolin to quinone methide 5
is the most energy-consuming step (Figure 4). Boltzmann
statistics predicted a final alphitonin−taxifolin equilibrium ratio
of 92:8, which is in excellent agreement with the experimental
result of a nearly complete conversion (Figure 6A,B).
Furthermore, the Gibbs free energies of 6−8 provided a
convincing explanation for the racemization without deuterium
incorporation, as the equilibrium populations are 76.8% for
alphitonin (8), 23.0% for the diketone 7, and only 0.2% for the
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EXPERIMENTAL SECTION
■
Standards and Materials. (±)-Taxifolin was obtained from
Sigma (lot 129H1722, Steinheim, Germany). Another sample of
(±)-taxifolin from Sigma (lot 015K4710) was identified as (+)-taxi-
folin. Solvents and reagents were of HPLC grade and obtained from J.
T. Baker (Griesheim, Germany) or Sigma.
General Experimental Procedures. 1H NMR spectra (500
MHz) were recorded on an Avance DRX 500 spectrometer (Bruker
BioSpin, Rheinstetten, Germany), and chemical shifts δ are given in
ppm referring to the signal center using the solvent peak for reference
(D2O: 4.79). IR spectra were obtained using a Bruker Tensor 27 FT-
IR spectrometer. Mass spectrometry was carried out on an API 2000
mass spectrometer (Applied Biosystems, Darmstadt, Germany).
Chromatographic analyses were performed using an HPLC system
consisting of a low-pressure mixing pump (model P580, Dionex,
Germering, Germany) equipped with a manual injection valve (model
8125, Rheodyne, Cotati, CA, USA) having a 20 μL (analytical) or 300
μL (semipreparative) sample loop and a UV or CD detector (model
UV-2075 or CD-2095, Jasco, Groß-Umstadt, Germany). Chromato-
grams were obtained using the Chromeleon (version 6.30, Dionex) or
ChromPass (version 1.8.6.1, Jasco) chromatography software.
Optimized HPLC conditions were applied as follows. Achiral
separation: 250 × 10.0 mm, 7 μm, C18-silica (Polygosil 60,
Macherey-Nagel, Duren, Germany), MeCN/H O/AcOH, 50/50/2,
̈
2
5.0 mL/min, 22 °C, 273 nm. Chiral separation: 150 × 4.6 mm, 5 μm,
cellulose tris(4-methylbenzoate) (OJ-RH, Daicel, Eschborn, Ger-
many), MeCN/H2O (pH 3, TFA), 10/90 (0 min), 10/90 (5 min),
20/80 (30 min), 70/30 (40 min), 10/90 (50 min), 0.5 mL/min, 35
°C, 273 nm. Semipreparative separation: 250 × 20.0 mm, 7 μM, C18-
silica (Polygosil 60, Macherey-Nagel), MeOH/H2O, 30/70, 5.0 mL/
min, 22 °C, 220 nm.
Chiral electrokinetic chromatography was carried out on a Beckman
P/ACE MDQ capillary electrophoretic system (Beckman Instruments,
Fullerton, CA, USA) equipped with a photo diode array detection
(DAD) system (scan range: 190−360 nm). All electropherograms
were processed using the Beckman 32Karat software version 7.0.
Uncoated fused-silica capillaries of 375 μm o.d. ×75 μm i.d. were
obtained from Beckman and cut to 50 cm (effective length 40 cm).
The following scheme was applied to wash the capillary (20 psi).
Before use: 0.25 M NaOH (20 min), then H2O (10 min); between
each run: 0.25 M NaOH (3 min), then H2O (2 min), 500 mM borate
buffer pH 9 (5 min); overnight storage: 0.25 M NaOH (5 min), then
H2O (10 min) and an air stream (3 min). The capillary temperature
was set to 20 °C, and the applied voltage was 18 kV. Samples were
injected hydrodynamically (0.3 psi) for 3 s. Chiral separation was
achieved using a 5 mM 2-hydroxypropyl-β-cyclodextrin solution in 500
mM borate buffer pH 9.
Figure 6. (A) Kinetic analysis of the thermal taxifolin−alphitonin
rearrangement at 115 °C monitored over a period of two weeks.
Achiral HPLC analysis of the conversion of racemic (±)-taxifolin
revealed a first-order mechanism (rate constant k = 3.60 × 10−6 s−1,
t0.5 = 3210 min; no separation of the dihydroquercetin stereoisomers;
the sum of the two AUCs was set at 100%). (B) Kinetics of the
enzyme-catalyzed conversion of racemic (±)-taxifolin to alphitonin at
22 °C in the presence of the partially purified enzyme (1.9 μg/mL)
from Eubacterium ramulus (first-order rate constant k = 7.26 × 10−4
s−1, t0.5 = 16 min; the sum of the five AUCs was set at 100%). (C)
Corresponding HPLC chromatograms at 0 min (a), 30 min (b), and
60 min (c) of the enzyme-catalyzed taxifolin−alphitonin rearrange-
ment showing the selective conversion of the (+)-taxifolin enantiomer.
Thermal Conversion of Taxifolin and Spectroscopic Char-
acterization of Alphitonin. (±)-Taxifolin (100 mg; Sigma, lot
129H1722) was placed in a 1 mL amber glass vial sealed with a septum
screw cap, and the vial was flushed with Ar to remove O2.
Subsequently, degassed H2O (1 mL) was added using a syringe to
replace the Ar, and the vial was placed inside a 10 mL glass reactor
(model TinyClave, Buchi, Flawil, Switzerland). The sample vial was
̈
submerged in degassed H2O (10 mL), and gallic acid (1 g) was added
as an external antioxidant. Finally, the glass reactor was flushed with Ar
and tightly sealed. The reaction apparatus was then heated to 115 °C
in an oil bath for 2 weeks, and samples were taken for in-process
control. Afterward, the solution was lyophilized to obtain a crude
product, of which a fraction (20.0 mg) was subjected to semi-
preparative HPLC to obtain (±)-alphitonin (11.2 mg) and quercetin
(6.0 mg). For chiral analysis, (+)-taxifolin (1 mg; Sigma, lot
015K4710) was reacted for a time period of 60 h as described
above. 1H NMR (D2O) δ 3.08 (d, 1H, 2J(H,H) = 13.9 Hz, CH2), 3.16
Table 3. Transition States Leading from the Diketone 7 to
Alphitonin (8) with One or Two Water Molecules for
a
Proton Relay
transition
state
O···C
(Å)
O−HA
O···HB
(Å)
O···CO
ΔG (kcal/mol)
(Å)
(deg)
1 H2O
2 H2O
2.86
2.34
2.632
2.533
0.984
0.979
1.990
1.904
97.002
96.767
2
4
(d, 1H, J(H,H) = 13.9 Hz, CH2), 5.86 (d, 1H, J(H,H) = 1.8 Hz, 5-
a
H), 5.96 (d, 1H, 4J(H,H) = 1.8 Hz, 7-H), 6.62 (dd, 1H, 3J(H,H) = 8.1
Results were obtained at the B3LYP/6-311G++(df,pd) level of
theory. Geometry data and energy barriers are given in comparison to
alphitonin (see Figure 5 for HA and HB).
4
3
Hz, J(H,H) = 2.0 Hz, 6′-H), 6.70 (d, 1H, J(H,H) = 8.1 Hz, 5′-H),
6.74 (d, 1H, J(H,H) = 2.0 Hz, 2′-H); IR (KBr, cm−1) 3422 (O−H),
4
1684 (CO), 1629 (CC); MS (ESI+) 305.3 ([C15H12O7 + H]+,
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16%), 286.9 ([C15H12O7 + H − H2O]+, 83%), 258.9 ([C15H12O7 + H
− H2O − CO]+, 100%), 230.9 ([C15H12O7 + H − H2O − 2 CO]+,
39%); MS (ESI−) 303.3 ([C15H12O7 − H]−, 89%), 285.1 ([C15H12O7
− H − H2O]−, 100%), 179.1 (5A− and 2,4B−, 7%), 177.3 (1,4B−, 12%),
151.1 (2,3A− and 2,3B−, 39%), 106.9 (1,4A−, 7%), 125.2 (2,4A−, 95%),
123.2 (5B−, 20%), 57.2 (6,7B−, 59%).
ACKNOWLEDGMENTS
■
The authors thank Torsten Bruhn (University of Wurzburg,
̈
Germany) and Henning Henschel (Linnæus University,
Sweden) for seminal support in quantum chemistry calculations
of CD spectra and transition states. We gratefully acknowledge
computational resources provided by the ARMINIUS cluster of
the Paderborn Center for Parallel Computing (University of
Paderborn, Germany). We also thank Andre Hutz (Jasco,
Groß-Umstadt, Germany) for the opportunity to use the CD
detector.
Enzymatic Transformation. The enzyme preparation was
obtained from cultures of Escherichia coli heterologously expressing
the taxifolin isomerase gene of E. ramulus (unpublished data). After
disruption of E. coli cells by sonication and subsequent centrifugation,
the resulting cell-free extract was fractionated by fast-performance
liquid chromatography using a DEAE-Sephacel column according to
the procedure described previously for cell extracts from E. ramulus.22
A fraction with high taxifolin-transforming activity was used for the
enzymatic assays. The assay contained 1165 μL of 50 mM K3PO4
buffer (pH 6.8) and 20 μL of the enzyme preparation (0.12 mg
protein/mL) and was performed at 22 °C. The reaction was started by
the addition of 65 μL of 1.2 mM (±)-taxifolin (Sigma, lot 129H1722)
in DMSO. Samples (50 μL) were taken every 10 min for 100 min and
immediately mixed with 50 μL of aqueous 10% MeCN acidified with
TFA to pH 1. Samples were submitted directly to chromatographic
analysis or were frozen using liquid nitrogen and stored at −20 °C.
Prediction of CD Spectra by Quantum Mechanical Calcu-
lations. Three-dimensional coordinates of (−)-2R,3S-epitaxifolin (4)
and (−)-2S,3S-taxifolin (2) were generated and subjected to a
preliminary MMFF94 force field minimization using the Chem3D
software. Using the ORCA program suite,48 a two-dimensional PES
scan was carried out at the B3LYP/TZVP level of theory applying a
COSMO model49 in methanol to simulate (i) a C-ring flip leading to
equatorial/axial inversion (φ 1 = C6−C7−C8−O9) and (ii) a full
rotation of the B−C-ring connecting bond (φ 2 = O9−C8−C10−
C11). A contour plot of the PES was prepared by a custom PERL
script using GnuPlot, and minimum energy conformers were identified
by visual inspection. These local minima were subjected to a free
optimization at the B3LYP/TZVP level of theory again applying a
COSMO methanol model, and final energies were obtained at the RI-
SCS-MP2/TZVP level for accurate Boltzmann statistics. CD spectra of
all local minima were then retrieved by a TDDFT/B3LYP/TZVP
approach and weighted according to their individual Boltzmann
population using SpecDis.
Calculations of Reaction Pathway Intermediates. Three-
dimensional coordinates of all reaction pathway intermediates and of
zwitterionic transition states with one or two H2O molecules for
proton relay were generated and subjected to a preliminary MMFF94
(intermediates) or MM2 (transition states) force field minimization
using the Chem3D software. Using the ORCA program suite,
minimum conformers of all intermediates were obtained at the
B3LYP/TZVP level of theory applying a COSMO water model,
followed by frequency calculations to obtain zero-point vibrational
energies at 25 and 115 °C. Using the Gaussian package38 at the
B3LYP/6-311G++(df,pd) level of theory, transition states with one or
two water molecules for proton relay were minimized with respect to
water orientation, while the oxonium scaffold was kept frozen. In
addition, structures of the diketone 7 and alphitonin (8) complex with
one or two H2O molecules connected by each transition state were
optimized and subjected to frequency calculations. A transition-state
search was carried out applying a STQN (QST3) approach at the
B3LYP/6-311G++(df,pd) computational level, followed by frequency
calculations to verify a single imaginary frequency and to obtain zero-
point vibrational energies for subsequent estimation of the
corresponding energy barrier.
̈
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AUTHOR INFORMATION
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Corresponding Author
*Tel: +49-89-3168-5033. Fax: +49-89-3168-5125. E-mail: paul.
elsinghorst@uni-bonn.de.
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