Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Production of 3,4-cis- and 3,4-trans-Leucocyanidin and Their Distinct MS/MS Fragmentation Patterns Jia-Rong Zhang, James Tolchard, Katell Bathany, Béatrice Langlois d’Estaintot, and Jean Chaudiere* Chimie et Biologie des Membranes et des Nano-objets (CBMN, UMR5248), Université 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
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 reductase (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-3ols, such as (−)-epicatechin, the most commonly occurring extension unit in proanthocyanidins. 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
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 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 antiinflammatory 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 leucoanthocyanidins, and to monitor their concentrations in complex Received: Revised: Accepted: Published: A
September 22, 2017 December 11, 2017 December 12, 2017 December 12, 2017 DOI: 10.1021/acs.jafc.7b04380 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Role of leucoanthocyanidin in the biosynthesis of anthocyanins and proanthocyanidins.
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 ANScatalyzed transformation of leucoanthocyanidins into anthocyanidins 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 stereoisomers of leucocyanidin to unambiguously clarify its enzymatic transformation into cyanidin or other products. In this Article, we describe the chemical synthesis of 3,4trans-leucocyanidin, its chemical epimerization to 3,4-cisleucocyanidin, the purification of each of these two stereoisomers by reverse-phase HPLC, and their structural assignment 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.
(≥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. 2.2. Chemical Synthesis of (+)-2,3-trans-3,4-cis-Leucocyanidin. 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-transleucocyanidin. 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-transLeucocyanidin to the (+)-2,3-trans-3,4-cis Isomer. (+)-2,3-trans3,4-cis-Leucocyanidin was obtained by acidic epimerization of the 3,4trans-leucocyanidin at the C4 position, using a modification of the
2. MATERIALS AND METHODS 2.1. Chemicals. (+)-2R,3R-Dihydroquercetin (DHQ) was purchased from ChemFaces (Wuhan, China). (+)-2R,3S-Catechin was obtained from Extrasynthèse (Lyon, France). NADPH (reduced nicotinamide adenine dinucleotide phosphate) tetrasodium salt B
DOI: 10.1021/acs.jafc.7b04380 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
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 (SigmaAldrich) 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).
method described by Kristiansen.21 A sample of the synthesized 3,4trans-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-transleucocyanidin. 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. 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 with Z-gradients. 2D 1H/1H total correlation spectroscopy (TOCSY) and 2D 1H/13C heteronuclear single-quantum correlation spectroscopy (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 Maugé et al.2 Approximately 5 mg of powders of 3,4-cis-leucocyanidin 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 3,4cis-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 wateracidified 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. 2.3.2. Spectrophotometric Quantification of (+)-2,3-trans-3,4-cisLeucocyanidin. 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-cisleucocyanidin (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. 2.4. MS/MS Analysis of the Two Leucocyanidin 3,4-Stereoisomers. 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
3. RESULTS 3.1. Chemical Synthesis of the (+)-2,3-trans-3,4-cisLeucocyanidin. 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
