Synthesis of the Main Red Wine Anthocyanin Metabolite: Malvidin-3-O-β-Glucuronide

Abstract

Anthocyanins are widely consumed in human and despite the health effects that have been attributed to them they appear to have low plasmatic concentration. This is in great part attributed to the lack of standards to allow an adequate assignment of the identity of the compounds detected that are often not the ones present in the original source. In this work, the chemical synthesis strategy of one of the glucuronide conjugates of the main anthocyanin present in red wine was developed. The purity and the identity of the metabolite were checked by HPLC-DAD, LC-MS, and NMR spectroscopy. It was confirmed that the compound obtained was malvidin-3-O-β-glucuronide, with the main MS² and MS³ fragments corresponding to the loss of glucuronic moiety [M – 176]⁺ m/z = 331 and methyl groups [M – 176 – 16]⁺ m/z = 315. The configuration and the position of the linkage between the aglycone and the glucuronyl residue was confirmed by NMR spectroscopy, considering the coupling constant between H-C1′′ and H-C2′′ and HMBC/NOESY connectivities.

Key words

anthocyanins - metabolites - red wine - glucuronidation - chemical synthesis

• References and Notes

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• 16 2-(2,3,4-Tri-O-acetyl-β-d-glucopyranuronic Acid Methyl Ester)-4′-hydroxy-3′,5′-dimethoxyacetophenone (3) α-Hydroxyacetosyringone (2, 280 mg, 1.32 mmol) and bromo-2,3,4-tri-O-acetyl-α-d-glucopyranuronic acid methyl ester (1.5 equiv) were mixed with an excess of Ag₂CO₃ (3 equiv) in anhydrous toluene (20 mL). The reaction was stirred at 100 °C for 2 h. The reaction was then filtered, the solvent removed under vacuum, and the product extracted with EtOAc and H₂O. The resulting dark oil was purified by column chromatography (CH₂Cl₂–MeOH, 20:1) giving a mixture of 3 and 4 (molar ratio of 1:0.2) as a pale yellow oil (206 mg, 26%). ESI-MS: m/z = 529 (3) [M + H]⁺ and 845 (4) [M + H]⁺.
• 17 Malvidin-3-O-β-glucuronide (6) The aldol condensation between 2,4-diacetoxy-6-hydroxybenzaldehyde (5, 34.1 mg, 0.14 mmol) and the mixture of 3 and 4 (1 equiv) was performed in dry EtOAc (5 mL), and anhydrous HCl (g) was bubbled through the solution. The reaction was stirred from 0 °C to r.t. overnight, and a red color gradually developed. Complete deacetylation was performed with KOH (3 equiv per OH) in H₂O–MeOH (1:1) during 15 min at r.t. under argon. After acidification to pH ~1 with 1 M HCl and MeOH removal, the aqueous fraction was extracted with EtOAc. The metabolite 6 was then purified by column chromatography loaded with RP-C18 silica gel (250 × 16 mm i.d.), and the pigment was eluted with 30% of aq MeOH solution acidified with HCl 2%. After MeOH evaporation and freeze-drying, compound 6 was obtained as a red solid (3.3 mg) which was used for NMR analyses. ¹H NMR (600.13 MHz, DMSO–TFA, 95:5): δ = 8.91 (s, 1 H, H-4C), 7.95 (s, 2 H, H-2′,6′B), 7.08 (d, J = 1.9 Hz, 1 H, H-8A), 6.76 (d, J = 1.9 Hz, 1 H, H-6A), 3.92 (s, 6 H, O(CH₃)₂); glucuronic acid: 5.56 (d, J = 7.5 Hz, 1 H, H-1′′), 4.02 (d, J = 9.5 Hz, 1 H, H-5′′), 3.47–3.40 (m, 3 H, H-2′′, H-3′′, H-4′′) ppm. ¹³C NMR (125.77 MHz, DMSO–TFA, 95:5): δ = 169.3 (C-7A), 161.7 (C-2C), 158.0 (C-5A), 156.3 (C-8aA), 148.6 (C-3′B, C-5′B), 144.8 (C-4′), 144.0 (C-3C), 136.2 (C-4C), 118.6 (C-1′B), 112.9 (C-4aA), 109.8 (C-2′B, C-6′B), 102.5 (C-6A), 94.9 (C-8A), 56.7 (O(CH₃)₂); glucuronic acid, 170.0 (COOH), 102.9 (C-1′′), 76.2 (C-5′′), 76.0 (C-3′′), 73.6 (C-4′′), 71.6 (C-2′′) ppm. LC-DAD/ESI-MS: full MS [M]⁺ m/z = 507; MS² [M – 176]⁺ m/z = 331; MS³ [M – 176 – 16]⁺ m/z = 315.
• 18 HPLC-DAD: HPLC analyses were performed on a Merck-Hitachi L-7100 (Merck, Darmstadt, Germany) apparatus with a 150 × 4.6 mm i.d. reverse-phase ODS C18 column (Merck, Darmstadt) at 25 °C; detection was carried out using a L-7450A diode array detector (DAD). The eluents were A: H₂O–HCOOH (90:10) and B: MeCN–H₂O–AcOH (80:19.5:0.5). The gradient consisted of 20–85% B for 70 min at a flow rate of 0.5 mL/min. The column was washed with 100% B during 10 min and then stabilized with the initial conditions during another 10 min.
• 19 LC-DAD/ESI-MS: LC-DAD/ESI/MS analyses were performed on a Finnigan Surveyor series liquid chromatograph equipped with Finnigan LCQ (Finnigan Corp., San Jose, CA, USA) mass detector and an API source using an ESI interface. The samples were analyzed on a reverse-phase column (150 × 4.6 mm, 5 μm, C18) at 25 °C using the same eluents, gradients, and flow rates referred for HPLC analysis. The capillary voltage was 4 V and the capillary temperature 275 °C. Spectra were recorded in positive and negative ion mode s between m/z = 120 and 1500. The mass spectrometer was programmed to do a series of three scans: a full mass (MS), a zoom scan of the most intense ion in the first scan (MS²), and a MS–MS of the most intense ion using relative collision energy of 30 and 60 (MS³).
• 20 ¹H NMR (600.13 MHz) and ¹³C NMR (125.77 MHz) spectra were recorded in DMSO–TFA (95:5) on a Bruker-Avance 600 spectrometer at 303 K and with TMS as an internal standard (chemical shifts (δ) in parts per million, coupling constants (J) in Hz). Multiplicities are recorded as singlets (s), doublets (d), and multiplets (m). ¹H chemical shifts were assigned using 2D NMR (COSY, NOESY) experiment while ¹³C resonances were assigned using 2D NMR techniques (gHMBC and gHSQC). The delay for the long-range C/H coupling constant was optimized to 7 Hz.
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