Bioinspired Synthesis and Physical-Chemical Properties of a New 10-Methylpyrano-4′-hydroxyflavylium Chloride Salt

Published as part of the Special Section 11th EuCheMS Organic Division Young Investigator Workshop

Abstract

A novel bioinspired 10-methylpyranoflavylium dye was synthesized. The dye was fully characterized by LC-DAD/ESI-MS and NMR 1D and 2D techniques, and its equilibrium constants towards pH variations were determined by UV–vis titration. These studies revealed the presence of three species (a flavylium cation AH⁺, a neutral quinoidal base A, and an anionic quinoidal base A^–) in the pH range 1–12, driven by the two pK _a values of the dye: pK _a1 = 6.8 ± 0.1 and pK _a2 = 10.8 ± 0.1. The first deprotonation of the dye occurs within the pH range for spoilage of many packed-food products, making the dye an excellent candidate for use as a pH sensor for real-time monitoring of the quality and freshness of foods.

Key words

methylpyranohydroxyflavylium - dye - pH sensor - food chemistry

• References and Notes

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• 14 2-(4-Hydroxyphenyl)-5-methylpyrano[4,3,2-de]chromen-1-ium Chloride (4) The dihydroxyflavylium chloride 3 (50 mg, 0.18 mmol) was dissolved in 10:90 acetone–H₂O, and the mixture was stirred at 37 °C and pH 2.9 for 3 d. The mixture was then extracted with Et₂O, and the aqueous fraction was purified by reverse-phase column chromatography [C₁₈ silica gel, MeOH–H₂O (10:90) acidified with 2% HCl]. The solvent was evaporated, and the product was freeze dried to give a dark-orange powder; yield: 17 mg (30%). ¹H NMR (600.13 MHz, 9:1 DMSO-d₆ –TFA): δ = 8.24 (d, J = 8.93 Hz, 2 H, H2′, H6′), 8.21 (t, J = 8.42 Hz, 1 H, H7), 7.88 (s, 1 H, H3), 7.87 or 7.73 (d, J = 4.21 Hz, 1 H, H8), 7.73 or 7.87 (d, J = 4.21 Hz, 1 H, H6), 7.09 (s, 1 H, H9), 7.06 (d, J = 8.90 Hz, 2 H, H3′, H5′), 2.65 (s, 3 H, CH₃). ¹³C NMR (125.77 MHz, 9:1 DMSO-d₆ –TFA): δ = 174.0 (C10), 170.0 (C2), 165.4 (C4′), 153.4 (C8a), 152.7 (C5), 137.4 (C7), 131.8 (C2′,C6′), 120.0 (C1′), 117.3 (C3′,C5′), 113.0 (C4a), 106.5 (C9), 102.4 (C3), 21.5 (CH₃). LC-DAD/ESI-MS: m/z = 277 [M⁺]. UV–vis: λ_max = 460 nm.
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• 19 HPLC-DAD analyses were performed on a Merck-Hitachi L-7100 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–HCO₂H (9:1) and B: CH₃CN. The gradient consisted of 0–35% B for 50 min at a flow rate of 0.5 mL/min. The column was washed with 100% B over 10 min and then stabilized to the initial conditions during another 10 min.
• 20 LC-DAD/ESI-MS: LC-DAD/ESI/MS analyses were performed on a Finnigan Surveyor series liquid chromatograph equipped with a Finnigan LCQ 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 as used for HPLC analysis (see Ref. 19). The capillary voltage was 4 V and the capillary temperature was 275 °C. Spectra were recorded in positive- and negative-ion modes between m/z = 120 and m/z = 1500. The mass spectrometer was programmed to carry out a series of three scans: a full mass scan, a zoom scan of the most intense ion in the first scan, and an MS–MS of the most intense ion using a relative collision energy of 30 and 60.
• 21 The ¹H NMR (600.13 MHz) and ¹³C NMR (125.77 MHz) spectra were recorded in 95:5 DMSO–TFA on a Bruker-Avance 600 spectrometer at 303 K, with TMS as an internal standard. ¹H chemical shifts were assigned by using 2D NMR (COSY, NOESY) experiments, whereas ¹³C resonances were assigned by using 2D NMR techniques (gHMBC and gHSQC). The delay for the long-range C–H coupling constant was optimized to 7 Hz.
• 22 UV-Vis Titration A 0.3 mM pigment stock solution was prepared in 0.1 M HCl in 75:25 EtOH–H₂O. A quartz cell was charged with Teorell and Stenhagen universal buffer²³ at pH 1 (1 mL), 0.1 M aq NaOH (1 mL), and the pigment stock solution (1 mL). The final concentration of the pigment was 0.1 mM in 25% EtOH. Titrations were performed up to pH 12 by the addition of small volumes of 1 M aq NaOH. Successive spectra were recorded immediately after the addition of the base in a Thermo Scientific Evolution Array UV–Visible spectrophotometer at 25 °C. All titrations were performed in triplicate. Final volumes were appropriately corrected. All pH measurements were made with a Radiometer Copenhagen PHM240 pH/ion meter. The fitting for pK _a determinations was carried out by using the Solver add-in program for Microsoft Excel.
• 23 Teorell and Stenhagen universal buffer was prepared by dissolving phosphoric acid (2.25 mL, 85% w/w), boric acid (3.54 g), monohydrated citric acid (7.00 g), and 1 M aq NaOH (343 mL) in distilled water to a volume of 1 L.
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