Production of 3,4-cis- and 3,4-trans-Leucocyanidin and Their Distinct MS/MS Fragmentation Patterns

Article abstract of DOI:10.1021/acs.jafc.7b04380

(+)-2,3-trans-3,4-cis-Leucocyanidin was produced by acidic epimerization of (+)-2,3-trans-3,4-trans-leucocyanidin synthesized by reduction of (+)-dihydroquercetin with NaBH₄, and structures of the two stereoisomers purified by C18- and phenyl-reverse-phase high-performance liquid chromatography (HPLC) were confirmed by NMR spectroscopy. We confirm that only 3,4-cis-leucocyanidin is used by leucoanthocyanidin reductase as substrate. The two stereoisomers are quite stable in aqueous solution at -20 °C. Characterization of the two stereoisomers was also performed using electrospray ionization tandem mass spectrometry (ESI-MS/MS), and we discuss here for the first time the corresponding MS/MS fragmentation pathways, which are clearly distinct. The main difference is that of the mode of dehydration of the 3,4-diol in positive ionization mode, which involves a loss of hydroxyl group at either C₃ or C₄ for the 3,4-cis isomer but only at C₃ for the 3,4-trans isomer. Tandem mass spectrometry therefore proves useful as a complementary methodology to NMR to identify each of the two stereoisomers.

Full text of DOI:10.1021/acs.jafc.7b04380

Article
pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Production of 3,4-cis- and 3,4-trans-Leucocyanidin and Their Distinct
MS/MS Fragmentation Patterns
Jia-Rong Zhang, James Tolchard, Katell Bathany, Beatrice Langlois d’Estaintot, and Jean Chaudiere*
́
́
Chimie et Biologie des Membranes et des Nano-objets (CBMN, UMR5248), Universite de Bordeaux, 33 608 Pessac, France
S
* Supporting Information
ABSTRACT: (+)-2,3-trans-3,4-cis-Leucocyanidin was produced by acidic epimerization of (+)-2,3-trans-3,4-trans-leucocyanidin
synthesized by reduction of (+)-dihydroquercetin with NaBH4, and structures of the two stereoisomers purified by C18- and
phenyl-reverse-phase high-performance liquid chromatography (HPLC) were confirmed by NMR spectroscopy. We confirm that
only 3,4-cis-leucocyanidin is used by leucoanthocyanidin reductase as substrate. The two stereoisomers are quite stable in
aqueous solution at −20 °C. Characterization of the two stereoisomers was also performed using electrospray ionization tandem
mass spectrometry (ESI-MS/MS), and we discuss here for the first time the corresponding MS/MS fragmentation pathways,
which are clearly distinct. The main difference is that of the mode of dehydration of the 3,4-diol in positive ionization mode,
which involves a loss of hydroxyl group at either C3 or C4 for the 3,4-cis isomer but only at C3 for the 3,4-trans isomer. Tandem
mass spectrometry therefore proves useful as a complementary methodology to NMR to identify each of the two stereoisomers.
KEYWORDS: leucocyanidin, NMR, leucoanthocyanidin reductase, reverse-phase HPLC, tandem mass spectrometry,
fragmentation pathways
This interest for their chromophoric properties has extended to
wine, as the presence of anthocyanins extracted from grapes
during fermentation can lead to the formation of monomeric
and polymeric pigments, which have a major contribution to
the stabilization of wine color.6−9
1. INTRODUCTION
Up to the present time, research studies on metabolism and
enzymatic production and transformation of one particular
subfamily of flavonoids, namely, leucoanthocyanidins, by
dihydroflavonol reductase (DFR),1 leucoanthocyanidin reduc-
tase (LAR),2 and anthocyanidin synthase (ANS)3 have been
hampered by the lack of commercially available standard
molecules and the lack of reliable protocol for their production,
as well as their nonambiguous analysis in the laboratory. For
years, the implicit idea was that such molecules were highly
unstable in aqueous solution, without clear proof that this is
indeed the case.
Leucoanthocyanidins are colorless flavan-3,4-diols related to
anthocyanins and proanthocyanidins (PAs), which are also
known as condensed tannins in the biosynthesis pathway of
flavonoids in plants. The biosynthetic pathway leading to
anthocyanins and proanthocyanidins branches off at the level of
leucoanthocyanidins,4 the common intermediates of these two
pathways (Figure 1). Leucoanthocyanidins arise from the
reduction of dihydroflavonols by DFR in vivo. Subsequently,
they are either reduced by LAR, the first enzyme in the PA
pathway, to produce (2R,3S)-flavan-3-ols, such as (+)-catechin,
the starter unit of most proanthocyanidins, or oxidized by ANS
(also known as leucoanthocyanidin dioxygenase, LDOX). ANS
is the first enzyme in the anthocyanin pathway and yields
anthocyanidins, which can be further glycosylated into
anthocyanins, the major pigments in plants. Alternatively,
anthocyanidins produced by ANS can also be reduced by
anthocyanidin reductase (ANR) to produce (2R,3R)-flavan-3-
ols, such as (−)-epicatechin, the most commonly occurring
extension unit in proanthocyanidins.
However, during the last two decades, a major shift of
interest has been observed, from color and flavor problems to
health benefits.10 Flavonoids have been shown to possess
antibacterial, antiviral, and antifungal properties that have been
highlighted as potential benefits of red wine consumption and
incentives for the development of winery byproducts.11
Flavonoids are also increasingly considered as useful
nutraceuticals in the prevention or treatment of cardiovascular
diseases,12 cancer, and inflammation, through their pro- or
antioxidative effects, or from their effects on signaling
mechanisms and epigenomic modifications.13 The anti-
inflammatory effects and other related health benefits of fruits
such as berries are believed to be due, at least in part, to their
high anthocyanin contents.14,15
With the increasing understanding of health benefits and
chemopreventive properties of anthocyanins and other
flavonoids, considerable effort has been invested into research
concerning the regulation of their biosynthesis in fruits of high
consumption,16 as well as in their pharmacokinetic properties
and bioavailability.17 There is therefore an increasing need to
develop reliable and nonambiguous analytical methods to
characterize the structures of flavonoids, including leucoantho-
cyanidins, and to monitor their concentrations in complex
Received: September 22, 2017
Revised: December 11, 2017
Accepted: December 12, 2017
Published: December 12, 2017
For many years, much of the research efforts on flavonoids
were focused on anthocyanidins and the anthocyanins derived
thereof, due to their importance as major pigments in plants.5
© XXXX American Chemical Society
A
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Figure 1. Role of leucoanthocyanidin in the biosynthesis of anthocyanins and proanthocyanidins.
(≥97% pure), sodium borohydride (NaBH4), methanol, and ethyl
acetate were purchased from Sigma-Aldrich. Ethanol absolute was
from Fisher Chemical (U.K.). All the organic solvents were of HPLC
grade.
biological samples containing mixtures of flavonoids, such as
fruits and vegetables or blood and urine.
Mass spectrometry has become a major tool in the analysis of
flavonoid structure because of its high sensitivity, its ability to
couple with liquid chromatography, and the powerful
techniques of tandem mass spectrometry.18,19 In the case of
leucoanthocyanidins, it is also important to shed light on their
stereochemistry as substrates of either LAR or ANS. The ANS-
catalyzed transformation of leucoanthocyanidins into antho-
cyanidins is the penultimate step in the biosynthesis of
anthocyanins and is generally considered as the main
physiological source of anthocyanidins in vivo. However,
ANS from Arabidopsis thaliana has been shown to transform
the natural stereoisomer of leucocyanidin into quercetin as the
major product instead of cyanidin, and this has raised doubts on
the role of leucoanthocyanidins as natural precursors of
anthocyanidins.3 Therefore, there is a need for pure stereo-
isomers of leucocyanidin to unambiguously clarify its enzymatic
transformation into cyanidin or other products.
In this Article, we describe the chemical synthesis of 3,4-
trans-leucocyanidin, its chemical epimerization to 3,4-cis-
leucocyanidin, the purification of each of these two stereo-
isomers by reverse-phase HPLC, and their structural assign-
ment by NMR. Additionally, we show that tandem mass
spectrometry (MS/MS) may also be used to distinguish the
two stereoisomers thanks to distinct fragmentation patterns.
2.2. Chemical Synthesis of (+)-2,3-trans-3,4-cis-Leucocyani-
din. 2.2.1. Preparation of (+)-2,3-trans-3,4-trans-Leucocyanidin
from (+)-DHQ. (+)-2,3-trans-3,4-trans-Leucocyanidin was prepared by
reduction of (+)-DHQ with NaBH4 according to the method
described by Stafford et al.,20 with some modifications. Five mg of
NaBH4 was added to 1 mL of absolute ethanol containing 10 mg of
(+)-DHQ in one batch, and the mixture was incubated for 2 h at room
temperature under vigorous stirring, resulting in a yellow solution.
After addition of 10 mL of H2O and immediate acidification to pH 4.0
by 20% acetic acid, the products were extracted with ethyl acetate (3 ×
5 mL). The ethyl acetate extract was washed with 0.5 M phosphate
buffer pH 8.0 (3 × 0.4 mL) and predominantly contained 3,4-trans-
leucocyanidin. The ethyl acetate extracts recovered from nine
production experiments were pooled. The resulting sample of ∼120
mL was rotary-evaporated at 20 °C to 30 mL, and 37.5 mL of
phosphate buffer pH 6.8 was then added. After complete evaporation
of ethyl acetate, the aqueous solution was frozen in liquid nitrogen and
lyophilized. The resulting white powder was dissolved in 7 mL of
methanol/H2O (50/50, v/v), and the 3,4-trans-leucocyanidin was
immediately purified by reverse-phase HPLC using a μBondapak
phenyl column (10 μm, 3.9 × 300 mm; Waters). This was carried out
at room temperature, and the 3,4-trans-leucocyanidin was eluted
isocratically with H2O under a flow rate of 2 mL/min; the fractions
were cut and collected into a flask based upon the 280 nm absorbance
monitoring. The collected aqueous sample of 3,4-trans-leucocyanidin
was subsequently lyophilized and recovered as a yellowish powder,
which was used to synthesize 3,4-cis-leucocyanidin as described below.
2.2.2. Chemical Conversion of the (+)-2,3-trans-3,4-trans-
Leucocyanidin to the (+)-2,3-trans-3,4-cis Isomer. (+)-2,3-trans-
3,4-cis-Leucocyanidin was obtained by acidic epimerization of the 3,4-
trans-leucocyanidin at the C4 position, using a modification of the
2. MATERIALS AND METHODS
2.1. Chemicals. (+)-2R,3R-Dihydroquercetin (DHQ) was pur-
chased from ChemFaces (Wuhan, China). (+)-2R,3S-Catechin was
obtained from Extrasynthes
nicotinamide adenine dinucleotide phosphate) tetrasodium salt
̀
e (Lyon, France). NADPH (reduced
B
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method described by Kristiansen.21 A sample of the synthesized 3,4-
trans-leucocyanidin in 2 mL of methanol was transferred to 50 mL of
0.1% acetic acid (aqueous solution, pH 3.2), and the mixture was
incubated at 40 °C for 80 min to epimerize the 3,4-trans-
leucocyanidin. The epimerization was monitored by reverse-phase
HPLC using a μBondapak C18 column (10 μm, 3.9 × 300 mm;
Waters) under a flow rate of 2 mL/min and a 30 min linear gradient
from 2 to 10% acetic acid (in water) at room temperature. After 80
min, the epimerization procedure was stopped by freezing the mixture
in liquid nitrogen, and the frozen sample was lyophilized. The resulting
yellowish powder was dissolved in 4 mL of methanol/H2O (50/50, v/
v), and 3,4-cis-leucocyanidin and its 3,4-trans isomer were purified by
reverse-phase HPLC using the μBondapak phenyl column with the
HPLC parameters already given in section 2.2.1. HPLC aqueous
fractions of 3,4-trans-leucocyanidin and 3,4-cis-leucocyanidin were
freeze-dried and recovered as yellowish and white powders,
respectively, and stored at −80 °C.
was used as desolvation gas at a temperature of 100 °C and a flow rate
of 350 L/h. A solution of [Glu1]-fibrinopeptide B human (Sigma-
Aldrich) at a concentration of 10 μg/mL in acetonitrile/0.1% aqueous
formic acid (v/v) was used for external calibration. MS mass spectra
were recorded with a scan time of 1 s in the mass range m/z 100−
1000. Tandem mass spectrometry analyses were carried out with argon
as collision gas using a collision energy of 3−10 eV and a collision cell
gas flow of 0.47 L/min. The full-scan MS/MS spectra were obtained
over an m/z range of 100−500. Data analysis was performed using the
MassLynx software (version 4.1).
3. RESULTS
3.1. Chemical Synthesis of the (+)-2,3-trans-3,4-cis-
Leucocyanidin. HPLC analysis of the reduction product of
(+)-DHQ showed two products that were expected to be the
two stereoisomers of leucocyanidin. On the basis of 280 nm
absorbance (Figure 2, in red), the product observed at 12.5 min
(peak 1) accounted for <2% of the total, whereas the product
observed at 21 min (peak 2) was by far the major one.
2.2.3. Characterization of Leucocyanidin Stereoisomers by NMR.
Dissolved in 500 μL of deuterated acetone-d6 were 1.3 mg of both
(+)-2,3-trans-3,4-cis-leucocyanidin and its 3,4-trans isomer. The
sample was adjusted to 100 μM TSP (trimethylsilylpropanoic acid)
in D2O, for use as an internal chemical shift reference. The sample was
transferred to a 5 mm NMR tube and incubated at 298 K for 10 min in
the spectrometer prior to measurement. All measurements were
carried out at 400 MHz, 298 K, using a Bruker AVANCE III
NANOBAY spectrometer equipped with a 5 mm BBFO SmartProbe
1
with Z-gradients. 2D H/1H total correlation spectroscopy (TOCSY)
1
and 2D H/13C heteronuclear single-quantum correlation spectrosco-
py (HSQC) spectra were used to confirm peak assignments.
2.3. Enzymatic Assay with LAR. LAR from Vitis vinifera (VvLAR,
untagged enzyme) was produced following the protocol described by
Mauge
́
et al.2 Approximately 5 mg of powders of 3,4-cis-leucocyanidin
Figure 2. Phenyl reverse-phase HPLC separation of 3,4-cis and 3,4-
trans isomers. In red: After the reduction of (+)-DHQ by NaBH4.
Peak 1 is 3,4-cis-leucocyanidin, and peak 2 is the 3,4-trans isomer. In
blue: After the acid-catalyzed epimerization of the major reduction
product (red peak 2). As will be shown later, peak 1 is 3,4-cis-
leucocyanidin and peak 2 is the 3,4-trans isomer.
and 3,4-trans isomer were dissolved in 2 and 9 mL of H2O,
respectively, and stored at −20 °C.
2.3.1. Reverse-Phase HPLC Monitoring of the Enzymatic
Reduction of (+)-2,3-trans-3,4-cis-Leucocyanidin. The reaction
mixture (1 mL) contained 150 μM NADPH, 0.025 mg/mL of 3,4-
cis-leucocyanidin, and 10 mM NaCl in 50 mM HEPES buffer, pH 6.5.
The reaction was initiated by the addition of 100 nM LAR and then
maintained for 20 min at 30 °C under gentle stirring. After incubation,
the reaction mixture was analyzed by reverse-phase HPLC using an
Atlantis C18 column (5 μm, 4.6 × 250 mm; Waters) with a 25 min
linear gradient from 20 to 90% B at 1 mL/min. Solvent A was water-
acidified with 0.1% (v/v) trifluoroacetic acid (TFA) and solvent B was
methanol-acidified with 0.1% TFA. The detection wavelength was 280
nm, and the column was held at 30 °C. One hundred μM (+)-catechin
in 50 mM HEPES buffer containing 10 mM NaCl was used as an
external standard.
During the incubation of 3,4-trans-leucocyanidin in diluted
acetic acid, three aliquots of the reaction mixture were removed
at 0, 40, and 80 min, respectively, and analyzed by reverse-
phase HPLC using the μBondapak C18 column to monitor the
epimerization process. As shown in Figure 3, the level of 3,4-
2.3.2. Spectrophotometric Quantification of (+)-2,3-trans-3,4-cis-
Leucocyanidin. LAR was used to catalyze the reductive transformation
of (+)-2,3-trans-3,4-cis-leucocyanidin into (+)-catechin in the presence
of NADPH. The initial concentration of NADPH was 150 μM, and
the NADPH consumption was monitored for 30 min by means of 340
nm absorbance measurement under magnetic stirring, in 50 mM
HEPES pH 7.5 at 30 °C, using a molar extinction coefficient of 6220
M−1 cm−1. Upon addition of 10 μL of aqueous solution of 3,4-cis-
leucocyanidin (0.025 mg/mL), the 340 nm absorbance was monitored
for 1 min, and the enzymatic reaction was then initiated by the
addition of 100 nM LAR.
Figure 3. C18 reverse-phase HPLC monitoring of the acidic
epimerization of 3,4-trans-leucocyanidin to 3,4-cis isomer. One
hundred μL of reaction mixture was analyzed at 0 (blue), 40
(green), and 80 min (red) after the addition of 3,4-trans-leucocyanidin.
2.4. MS/MS Analysis of the Two Leucocyanidin 3,4-Stereo-
isomers. Stereoisomers of leucocyanidin were characterized by
electrospray ionization tandem mass spectrometry (ESI-MS/MS).
Mass spectrometry was performed on a Q-Tof Premier mass
spectrometer (Waters) equipped with an electrospray ionization
source (ESI) and a time-of-flight (TOF) analyzer. The solutions of
stereoisomers were infused with a syringe pump at a flow rate of 5 μL/
min into the ESI source. MS analyses were performed in positive ion
mode under the following conditions: the ESI source temperature was
100 °C, the source capillary voltage was set at 3 kV, the sampling cone
voltage was 30 V, and the cone gas flow was set at 50 L/h. Nitrogen
trans-leucocyanidin (peak 2, 10.6 min) declined and an increase
in 3,4-cis-leucocyanidin (peak 1, 7 min) occurred simulta-
neously. After 80 min of incubation, concentrations of 3,4-cis-
leucocyanidin and 3,4-trans isomer were approximately the
same. Longer incubation times caused a decline in the two
stereoisomers of leucocyanidin. Finally, the two stereoisomers
of leucocyanidin were purified by reverse-phase HPLC using
the μBondapak phenyl column (Figure 2, in blue).
C
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Figure 4. 1D 1H NMR of 2.6 mg·mL−1 of 3,4-cis-leucocyanidin (blue) and 3,4-trans isomer (red) dissolved in deuterated acetone-d6, acquired at 400
MHz and 298 K.
The two structures of 3,4-cis- and 3,4-trans-leucocyanidin
were confirmed by NMR spectroscopy. The 1D H NMR
Table 1. NMR Chemical Shifts and Coupling Constants of
Leucocyanidins
1
spectrum of (+)-2,3-trans-3,4-cis-leucocyanidin is presented in
chemical shifts
a
1H coupling
b
leucocyanidin
position
1H/13C (δ, ppm)
constants (J, Hz)
Figure 4 (in blue). It is highly comparable to that previously
published,21 in spite of incorrect original H chemical shift
1
H/C-2
H/C-3
H/C-4
H/C-6
H/C-8
H/C-2′
H/C-5′
H/C-6′
4.84/79.43
3.84/73.10
4.85/64.43
5.84/96.92
6.01/98.05
6.93/117.40
6.79/117.32
6.79/122.23
9.60 (d)
3.84, 9.28 (dd)
4.04 (d)
2.24 (d)
2.28 (d)
1.60 (d)
-
assignments for the H2′, H5′, and H6′ aromatic moieties at
that time. Furthermore, the J2,3 and J3,4 coupling constants are
in agreement with a 2,3-trans-3,4-cis configuration, as
previously stated21,22 (Table 1).
3,4-cis-
leucocyanidin
The NMR spectrum of (+)-2,3-trans-3,4-trans-leucocyanidin
(Figure 4, in red), is somewhat similar to that of the 3,4-cis
isomer but has markedly different H2 (upfield) and H4
(downfield) chemical shifts. These distinct chemical shifts are
expected in the 2,3-trans-3,4-trans configuration, where the
greater distance between H2 and O4, as well as the increased
proximity between H4 and O3, would induce chemical shift
shielding and deshielding, respectively. The increased 3,4-trans
J3,4 coupling (7.96 Hz, Table 1) is also indicative of a 2,3-trans-
3,4-trans configuration, as previously published.23 The stereo-
isomerism of each compound was further probed by the use of
1D selective 1H nuclear Overhauser effect spectroscopy
(NOESY) spectra, where data were in agreement with the
expected H3 to H2 and H4 internuclear proximities for each
configuration (data not shown).
-
H/C-2
H/C-3
H/C-4
H/C-6
H/C-8
H/C-2′
H/C-5′
H/C-6′
4.59/83.78
3.85/75.80
4.96/74.63
5.82/97.24
5.95/99.02
6.93/117.56
6.79/117.40
6.79/122.48
10.00 (d)
8.00, 9.96 (dd)
7.96 (d)
2.28 (d)
2.32 (d)
1.00 (d)
-
3,4-trans-
leucocyanidin
-
a
1H and 13C chemical shifts were referenced to the internal acetone
solvent. d = doublet, dd = doublet of doublets.
b
3.2. Enzymatic Assay with LAR. 3.2.1. Use of LAR To
Discriminate between 3,4-cis-Leucocyanidin and 3,4-trans
Isomer. Before the enzyme assay with LAR, the stock solutions
of these two stereoisomers were analyzed by reverse-phase
HPLC using the μBondapak C18 column to check their purity.
As shown in Figure 5, the final sample of 3,4-cis-leucocyanidin
(peak 1 in Figure 5A) contained ∼5% 3,4-trans-leucocyanidin
(peak 2 in Figure 5A), whereas the final sample of 3,4-trans-
leucocyanidin (peak 2 in Figure 5B) contained ∼13% 3,4-cis-
leucocyanidin (peak 1 in Figure 5B).
Enzymatic assays were carried out with recombinant LAR
employing either 3,4-cis- or 3,4-trans-leucocyanidin as substrate.
As shown in Figure 6, a single product (red peak 1′, 10.3 min)
was observed using 3,4-cis-leucocyanidin as substrate and
assigned to (+)-catechin using a commercial standard (blue
peak 1, 10.1 min). The small chromatogram inserted in Figure
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sample was also quantified with LAR, and its percentage of loss
calculated from enzymatic quantification was compared with
that obtained from chromatographic data.
After 70 days of storage at −20 °C, there was no significant
change in 280 nm absorbance for the two stereoisomers
(Figure S2). The percentages of loss of 3,4-cis-leucocyanidin
calculated from HPLC analysis and from enzymatic quantifi-
cation were 5.5% and 5.8%, respectively, which suggests that
the HPLC quantification is reliable. With 3,4-trans-leucocyani-
din, which could not be quantified with LAR, the percentage of
loss was 3.8% based on HPLC analysis. Our data suggest that
the two stereoisomers of leucocyanidin are quite stable in H2O
at −20 °C.
3.4. MS/MS Analysis of the Two Stereoisomers of
Leucocyanidin. The fragmentation of the [M + H]+ ion with
m/z 307 corresponding to the 3,4-cis-leucocyanidin produced 6
major fragment ions with m/z 127, 139, 155, 163, 271, and 289
observed on the MS/MS spectrum (Figure 7A), whereas the
Figure 5. C18 reverse-phase HPLC analysis of final samples of 3,4-cis-
leucocyanidin (A) and 3,4-trans-leucocyanidin (B).
Figure 6. C18 reverse-phase HPLC analysis of LAR product of 3,4-cis-
leucocyanidin. Red peak 1′ is the only enzymatic product, which was
assigned to (+)-catechin, and blue peak 1 is the commercial standard
of (+)-catechin (100 μM). The polar products observed between 4
and 6 min are NADPH and its oxidation product.
Figure 7. Positive ion ESI-MS/MS spectra of the ion with m/z 307
corresponding to the [M + H]+ ion derived from 3,4-cis-leucocyanidin
(A) and 3,4-trans-leucocyanidin (B).
6 shows the result of the control experiment (red) performed
with 3,4-cis-leucocyanidin in the absence of LAR, in which red
peak 1 is 3,4-cis-leucocyanidin, with no product observed at the
retention time of the commercial standard of (+)-catechin
(blue peak 2, 10.1 min). This means that (+)-catechin (red
peak 1′) is obtained only in the presence of LAR. With 3,4-
trans-leucocyanidin, no product was observed (data not
shown), implying that LAR only accepts the 3,4-cis isomer as
substrate.
3.2.2. Quantification of 3,4-cis-Leucocyanidin by Means
of LAR. In this enzymatic reaction, 1 equiv of NADPH is
consumed for each 3,4-cis-leucocyanidin transformed into
(+)-catechin. A fall in the 340 nm absorbance of NADPH is
immediately observed after the addition of LAR, and this
absorbance is stabilized by 17 min, implying that the initially
added 3,4-cis-leucocyanidin has been completely consumed
(Figure S1, Supporting Information). By 30 min, the total
absorbance decrease of NADPH was 0.48 and that of the
control experiment was 0.08. Therefore, the calculated
concentration of 3,4-cis-leucocyanidin is 6.5 mM based on the
molar extinction coefficient of NADPH.
fragmentation of the [M + H]+ ion with m/z 307 derived from
3,4-trans-leucocyanidin produced 5 major fragment ions with
m/z 127, 155, 163, 271, and 289 (Figure 7B). In addition, there
is an ion with m/z 327, which is most likely a single-charge
potassium adduct ion, formed by exchange of proton for
potassium in fragment ion with m/z 289. One can see that
there are two major differences between the MS/MS spectra of
the two stereoisomers: the presence of fragment ion with m/z
139 in the MS/MS spectrum of 3,4-cis-leucocyanidin only, and
a signal-intensity inversion between their common fragment
ions with m/z 155 and 163. These data show that the two
stereoisomers have distinct fragmentation pathways that have
not been described before.
4. DISCUSSION
The MS/MS fragmentation pathways of catechin based on the
fragment ions derived from retro-Diels−Alder (RDA) fission,
heterocyclic ring (HRF) fission, and benzofuran forming (BFF)
fission were detailed by Li and Deinzer.24 On the basis of their
study, the MS/MS fragmentation pathways of the two
stereoisomers of leucocyanidin were also established from a
combination of three distinct fragmentation patterns including
RDA, HRF, and an additional quinone methide (QM) fission.
3.3. Stability of the Two Stereoisomers of Leucocya-
nidin. The aqueous solutions of the two stereoisomers were
stored at −20 °C for 70 days to check their stability. Samples
were analyzed by reverse-phase HPLC using the μBondapak
C18 column. In addition, the stored 3,4-cis-leucocyanidin
E
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Figure 8. Postulated MS/MS fragmentation pathways of 3,4-cis-leucocyanidin (A) and 3,4-trans-leucocyanidin (B), based on three distinct
fragmentation patterns: RDA, HRF, and QM.
Three different water eliminations based on two different
protonation positions of the two stereoisomers of leucocyanidin
were envisaged (Figure 8), and we assume that the specific MS/
MS fragmentation pattern of 3,4-cis-leucocyanidin is due to
water elimination at C3−C4 by loss of the C4 hydroxyl group, as
described in Figure 8A. The elimination of water by QM fission
could take place only with 3,4-cis-leucocyanidin 1 protonated at
C4 hydroxyl group, and ion 1.1 with m/z 289 would be the only
source of fragment ion 1.2 with m/z 271. As shown on the right
side of Figure 8A, 3,4-cis-leucocyanidin 2 protonated on the
oxygen atom of ring C would only undergo RDA and HRF
fissions. Ion 2.1 with m/z 155 would result from direct RDA
fission (307−152 Da). Water elimination also occurs from the
precursor ion 2 with m/z 307, with elimination of a hydroxyl
group at C3 or C4. The elimination of water at C3−C4 by loss of
the C3 hydroxyl group would yield ion 2.2 with m/z 289, from
which fragment ion 2.4 with m/z 163 could be formed by HRF
fission (289−126 Da), whereas the elimination of water at C3−
C4 by loss of the C4 hydroxyl group would yield ion 2.5 with
m/z 289, which is the only source of fragment ion 2.7 with m/z
139.
According to the MS/MS spectra of Figure 7, the major
differences between the spectra of the two stereoisomers bear
on two fragment ions: m/z 139 and m/z 155. The fragment ion
with m/z 139 only observed on the MS/MS spectrum of 3,4-
cis-leucocyanidin was recorded as a major fragment ion, which
indicates that 3,4-cis-leucocyanidin was mostly protonated on
the oxygen atom of ring C (2, Figure 8A). Removal of the C4
hydroxyl group by dehydration of the protonated 3,4-cis-
leucocyanidin (2, Figure 8A) yields an abundant fragment ion
with m/z 289 (2.5, Figure 8A), which eventually gives a
fragment ion with m/z 139 (2.7, Figure 8A). On the other
hand, the strong signal intensity of the fragment ion with m/z
155 observed with 3,4-trans-leucocyanidin indicates that 3,4-
trans-leucocyanidin was also mainly protonated on the oxygen
atom of ring C (2, Figure 8B), but the latter was mainly
fragmented by direct RDA fission instead of water elimination,
to yield an abundant fragment ion with m/z 155 (2.1, Figure
8B).
A final comment may be made on the ion with m/z 327
(Figure 7), which is assumed to be a potassium adduct of
fragment ion m/z 289. The signal intensity of the ion with m/z
327 is indeed much higher with the 3,4-cis isomer than with the
3,4-trans isomer, which suggests that more fragment ion with
m/z 289 has been produced with the former. This is in
agreement with the existence of two dehydration pathways at
C3−C4 for the 3,4-cis isomer (loss of OH at C3 or C4)
compared with only one for the 3,4-trans isomer (loss of OH at
C3).
As for the MS/MS fragmentation pathway of 3,4-trans-
leucocyanidin (Figure 8B), all fragment ions with m/z 289 were
obtained from water elimination either by QM fission 1.1 from
the 3,4-trans-leucocyanidin 1 protonated at C4 hydroxyl group
or by removal of the C3 hydroxyl group 2.2 from the 3,4-trans-
leucocyanidin 2 protonated on the oxygen atom of ring C, but
water elimination by loss of the C4 hydroxyl group was not
observed.
One may assume that, in the 3,4-trans isomer, protonation of
the benzylic hydroxyl group at C4 is much more difficult than
F
DOI: 10.1021/acs.jafc.7b04380
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
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that of the hydroxyl group at C3, whereas in the 3,4-cis isomer,
any proton added to the hydroxyl group at C3 would be shared
with the hydroxyl group neighbor at C4.
In conclusion, the MS/MS characterization of the two
stereoisomers of leucocyanidin has been reported for the first
time, to the best of our knowledge, as well as the MS/MS
fragmentation pathways of these two stereoisomers. The
identification of the two stereoisomers of leucocyanidin by
MS/MS was achieved based on their distinct MS/MS
fragmentation pathways, a new approach that is complementary
to NMR, wherein the unambiguous distinction of stereoisomers
can be difficult or impossible. Furthermore, the stability of each
of these two stereoisomers in aqueous solution was also
detailed. Our results obtained from both the enzymatic reaction
catalyzed by VvLAR and the reverse-phase HPLC analysis
showed that the two stereoisomers could be stored in water at
−20 °C for 70 days with <6% loss. With the ability to
synthesize the pure stereoisomers of leucocyanidin, clean
kinetic studies of the reductive transformation of 3,4-cis-
leucocyanidin by VvLAR could be carried out, as well as studies
of the conversion of leucocyanidin to cyanidin or quercetin by
VvANS.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.7b04380.
(12) Rodriguez-Mateos, A.; Heiss, C.; Borges, G.; Crozier, A. Berry
(poly)phenols and cardiovascular health. J. Agric. Food Chem. 2014, 62,
3842−3851.
Spectrophotometric quantification of the 3,4-cis-leuco-
cyanidin with LAR and C18 reverse-phase HPLC
analysis of the stability of the two stereoisomers of
leucocyanidin (PDF)
(13) Chu, K. O.; Chan, S. O.; Pang, C. P.; Wang, C. C. Pro-oxidative
and antioxidative controls and signaling modification of polyphenolic
phytochemicals: contribution to health promotion and disease
prevention? J. Agric. Food Chem. 2014, 62, 4026−4038.
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(15) Joseph, S. V.; Edirisinghe, I.; Burton-Freeman, B. M. Berries:
anti-inflammatory effects in humans. J. Agric. Food Chem. 2014, 62,
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +33 (0)6 63 07 53 48. Fax: +33 (0)5 40 00 24 38. E-
mail: jean.chaudiere@u-bordeaux.fr.
■
ORCID
Jean Chaudiere: 0000-0002-9420-1473
(16) Lo Piero, A. R. The State of the art in biosynthesis of
anthocyanins and its regulation in pigmented sweet oranges [(Citrus
sinensis) L. Osbeck]. J. Agric. Food Chem. 2015, 63, 4031−4041.
(17) Sandhu, A. K.; Huang, Y.; Xiao, D.; Park, E.; Edirisinghe, I.;
Burton-Freeman, B. Pharmacokinetic characterization and bioavail-
ability of strawberry anthocyanins relative to meal intake. J. Agric. Food
Chem. 2016, 64, 4891−4899.
Funding
J.-R.Z. gratefully acknowledges a three-year funding Ph.D.
scholarship from the department of Bioengineering (Qilu
University of Technology, China).
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We would like to thank our colleague Claude Manigand for his
technical assistance with the HPLC analysis. This work has
benefited from the facilities and expertise of the Biophysical and
Structural Chemistry platform (BPCS) at IECB (UMS3033/
US001).
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