Materials
l-Glucose and β-glucosidase (from Almond, 121 U/mg) were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan) and Oriental Yeast Co. (Tokyo, Japan), respectively. All other chemicals were purchased from commercial sources and used without further purification unless otherwise noted.
Measurements
1H and 13C NMR spectra were recorded on a Varian INOVA300 MHz FT-NMR (300 MHz) spectrometer (Agilent Technologies, Santa Clara, CA, USA) using tetramethylsilane as an internal standard in DMSO-d6 for compounds 1L and 1D or CDCl3 for others. Chemical shift (δ) and coupling constant (J) are given in ppm (parts per million) and Hz, respectively. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) measurements were recorded on a Bruker MALDI-TOF MS REFLEX III (Bruker, Billerica, MA, USA) in the positive and linear ion modes. A nitrogen laser was used for the ionization of the samples. All spectra were obtained using 2,5-dihydroxybenzoic acid as a matrix. Specific rotations were recorded on a JASCO P-2200 polarimeter (JASCO, Hachioji, Japan) in H2O for compounds 1L and 1D or CHCl3 for others, and were determined as the average values of five measurements. Melting points (m.p.) were measured in a micro-melting point apparatus (Yanagimoto Seisakusho, Kyoto, Japan). UV–Vis spectra were recorded on a JASCO V-560 spectrophotometer (JASCO).
Preparation of l-coniferin (1L)
Compound 1L was prepared from l-glucose (2L) according to the conventional method for compound 1D [10] as shown in Fig. 2.
l-Glucose pentaacetate (3L)
Compound 2L (5 g, 27.8 mmol) and CH3COONa (2.5 g, 30.5 mmol) were suspended in Ac2O (25 mL). The suspension was stirred at 80 °C for 3.5 h, cooled to ambient temperature and extracted with EtOAc. The organic layer was washed with a saturated NaHCO3 solution, distilled water, and brine, dried over Na2SO4 and evaporated to give a colorless residue. The residue was recrystallized from EtOH to give compound 3L (9.63 g, 88.9% yield).
Compound 3L: \(\left[ \alpha \right]_{D}^{25}\) = − 20.7° (c = 1.03, in CHCl3); 1H and 13C NMR spectra data of compound 3L were in agreement with the published data of l-glucose pentaacetate [11]; MALDI-TOF MS: m/z calcd. for [M+Na]+ C16H22NaO11: 413.34, found: 413.56.
2,3,4,6-Tetra-O-acetyl-α-l-glucopyranosyl bromide (4L)
Compound 3L (1000 mg, 2.56 mmol) was dissolved in CH2Cl2 (2 mL). 33%-HBr in AcOH (2.5 mL, 14.4 mmol) was added to the solution at 0 °C. After stirring the reaction solution at ambient temperature for 2 h, distilled water (10 mL) was added to the solution at 0 °C. The reaction mixture was extracted with EtOAc. The organic layer was washed with distilled water, a saturated NaHCO3 solution, and brine, dried over Na2SO4 and evaporated to give a colorless residue. The residue was recrystallized from EtOH to give compound 4L (845 mg, 80.2% yield).
Compound 4L: \(\left[ \alpha \right]_{D}^{25}\) = − 194.3° (c = 1.04, in CHCl3); 1H NMR (CDCl3): δ 6.62 (d, 1H, J = 4.2, H-1), 5.56 (t, 1H, J = 9.8, H-3), 5.16 (t, 1H, J = 9.8, H-4), 4.84 (dd, 1H, J = 9.8, 4.2, H-2), 4.33 (dd, 1H, J = 14.1, 4.2, H-6a), 4.30 (ddd, J = 9.8, 4.2, 1.5, H-5), 4.13 (dd, 1H, J = 14.1, 1.5, H-6b), 2.11, 2.10, 2.06, 2.04 (s, 3H, acetyl CH3); 13C NMR (75 MHz, CDCl3): δ 170.6, 170.0, 169.9, 169.6 (acetyl C=O), 86.7 (C-1), 72.2 (C-5), 70.7 (C-2), 70.3 (C-3), 67.3 (C-4), 61.0 (C-6), 20.8, 20.8, 20.7, 20.7 (acetyl CH3); MALDI-TOF MS: m/z calcd. for [M+Na]+ C14H19BrNaO9: 433.20, found: 434.99.
(4-Formyl-2-methoxy)phenyl 2,3,4,6-tetra-O-acetyl-β-l-glucopyranoside (5L)
Compound 4L: (700 mg, 1.65 mmol) and vanillin (250 mg, 1.65 mmol) were dissolved in quinoline (4.5 mL). Ag2O (390 mg) was added to the solution at 0 °C. The reaction mixture was stirred at ambient temperature for 1.5 h, filtered through Celite (535RVS, Nacalai Tesque (Kyoto, Japan)), and extracted with EtOAc. The organic layer was washed with a 1 M HCl solution, a saturated NaHCO3 solution, and brine, dried over Na2SO4 and evaporated to give a light brown residue. The residue was recrystallized from EtOH to give compound 5L (599 mg, 72.9% yield).
Compound 5L: \(\left[ \alpha \right]_{D}^{25}\) = + 42.9° (c = 0.53, in CHCl3); 1H NMR (300 MHz, CDCl3): δ 9.90 (s, 1H, CHO), 7.44 (d, 1H, J = 2.1, H-2′), 7.42 (dd, 1H, J = 8.7, 2.1, H-6′), 7.22 (d, 1H, J = 8.7, H-5′), 5.37–5.27 (m, 2H, H-2, H-3), 5.22–5.15 (m, 1H, H-4), 5.11 (d, 1H, J = 7.8, H-1), 4.28 (dd, 1H, J = 12.3, 5.4, 1H, H-6a), 4.19 (dd, 1H, J = 12.3, 2.7, H-6b), 3.90 (s, 3H, OCH3), 3.85 (ddd, 1H, J = 9.8, 5.4, 2.7, H-5), 2.08, 2.08, 2.05, 2.05 (s, 3H, acetyl CH3); 13C NMR (75 MHz, CDCl3): δ 191.1 (C-α), 170.7, 170.4, 169.5, 169.4 (acetyl C=O), 151.2 (C-4′), 151.1 (C-3′), 133.0 (C-1′), 125.5 (C-6′), 118.3 (C-5′), 110.8 (C-2′), 99.9 (C-1), 72.5 (C-3), 72.4 (C-5), 71.1 (C-2), 68.4 (C-4), 62.0 (C-6), 56.2 (OCH3), 20.9, 20.9, 20.8, 20.8 (acetyl CH3); MALDI-TOF MS: m/z calcd. for [M+Na]+ C22H26NaO12: 505.44, found: 505.35.
(4-Ethoxycarbonyl-2-methoxy)phenyl 2,3,4,6-tetra-O-acetyl-β-l-glucopyranoside (6L)
Compound 5L (1000 mg, 2.07 mmol) and ethyl malonic acid (520 mg, 3.96 mmol) were dissolved in pyridine (16.5 mL). After the addition of piperidine (0.275 mL, 2.78 mmol), the reaction solution was stirred at 100 °C for 1.5 h and concentrated by azeotrope distillation with EtOH to give a colorless residue. The residue was recrystallized from EtOH to give compound 6L (979 mg, 85.9% yield).
Compound 6L: \(\left[ \alpha \right]_{D}^{25}\) = + 27.2° (c = 0.56, in CHCl3); 1H NMR (300 MHz, CDCl3): δ 7.62 (d, 1H, J = 16.2, H-α), 7.10 (d, 1H, J = 8.4, H-5′), 7.07 (d, 1H, J = 1.5, H-2′), 7.05 (dd, 1H, J = 8.4, 1.5, H-6′), 6.35 (d, 1H, J = 16.2, H-β), 5.40–5.26 (m, 2H, H-2, H-3), 5.20–5.13 (m, 1H, H-4), 5.02–5.00 (m, 1H, H-1), 4.28 (dd, 1H, J = 12.5, 5.1, H-6a), 4.26 (dd, 2H, J = 14.1, 7.2, CH2), 4.17 (dd, 1H, J = 12.5, 2.4, H-6b), 3.85 (s, 3H, OCH3), 3.80 (ddd, 1H, J = 10.2, 5.1, 2.4, H-5), 2.08, 2.05, 2.05, 2.04 (s, 3H, acetyl CH3), 1.34 (t, 3H, J = 7.2, ethyl CH3); 13C NMR (75 MHz, CDCl3): δ 170.5, 170.3, 169.4, 169.3 (acetyl C=O), 166.9 (C-ɤ), 150.7 (C-3′), 147.7 (C-4′), 143.9 (C-α), 131.0 (C-1′), 121.6 (C-6′), 119.5 (C-5′), 117.6 (C-β), 111.3 (C-2′), 100.3 (C-1), 72.4 (C-3), 72.0 (C-5), 71.0 (C-2), 68.3 (C-4), 61.8 (C-6), 60.5 (ethyl CH2), 56.0 (OCH3), 20.7, 20.6, 20.6, 20.6 (acetyl CH3), 14.3 (ethyl CH3); MALDI-TOF MS: m/z calcd. for [M+Na]+ C26H32NaO13: 575.53, found: 575.57.
(4-(3-Hydroxy-2-propenyl)-2-methoxy)phenyl β-l-glucopyranoside (l-coniferin) (1L)
Compound 6L (900 mg, 1.63 mmol) was dissolved in toluene (22 mL). The 1.01 mol/L of DIBAL-H in toluene (15 mL, 15.1 mmol) was added dropwise to the solution at 0 °C for 10 min. The reaction mixture was stirred at 0 °C for 1 h and EtOH (20 mL) was added slowly to the mixture. The reaction mixture was stirred at 0 °C for another 30 min, concentrated and filtered with hot water. The filtrate was concentrated by azeotrope distillation with EtOH to give a colorless residue. The residue was purified by preparative thin-layer chromatography using a silica gel plate (silica gel 60F254, 2-mm thickness, Merck, Darmstadt, Germany) developed with 20% MeOH/CH2Cl2 (v/v) and recrystallized from water three times to give compound 1L (295 mg, 49.1% yield).
Compound 1L: m.p.: 179–182 °C; \(\left[ \alpha \right]_{D}^{25}\) = + 65.8° (c = 0.5, in CHCl3); 1H NMR (300 MHz, DMSO-d6): δ 7.06 (d, 1H, J = 1.8, H-2′), 7.02 (d, 1H, J = 8.4, H-5′), 6.90 (dd, 1H, J = 8.4, 1.8, H-6′), 6.48 (d, 1H, J = 15.9, H-α), 6.28 (dt, 1H, J = 15.9, 5.1, H-β), 5.25 (d, 1H, J = 4.8, OH-3), 5.10 (d, 1H, J = 4.8, OH-2), 5.04 (d, 1H, J = 4.8, OH-4), 4.89 (d, 1H, J = 8.1, H-1), 4.85 (d, 1H, J = 5.4, OH-γ), 4.57 (t, 1H, J = 5.4, OH-6), 4.10 (dd, 1H, J = 5.1, 1.2, H-ɤa), 4.09 (dd, 1H, J = 5.1, 1.2, H-ɤb), 3.79 (s, 3H, OCH3), 3.68–3.65 (m, 1H, H-6a), 3.52–3.17 (m, 5H, H-2, H-3, H-4, H-5, H-6b); 13C NMR (75 MHz, DMSO-d6): δ 149.4 (C-3′), 146.4 (C-4′), 131.4 (C-1′), 129.4 (C-β), 128.9 (C-α), 119.5 (C-6′), 115.6 (C-5′), 110.2 (C-2′), 100.4 (C-1), 77.4 (C-3), 77.3 (C-5), 73.7 (C-2), 70.7 (C-4), 62.1 (C-ɤ), 61.1 (C-6), 56.0 (OCH3); MALDI-TOF MS: m/z calcd. for [M+Na]+ C16H22NaO8: 365.34, found: 365.30.
Preparation of d-coniferin (1D)
Compound 1D was also prepared by the conventional method as shown in Fig. 2a [10]. Compound 3D: \(\left[ \alpha \right]_{D}^{25}\) = + 21.4° (c = 1.34, in CHCl3); 4D: \(\left[ \alpha \right]_{D}^{25}\) = + 193.5° (c = 1.13, in CHCl3), 5D: \(\left[ \alpha \right]_{D}^{25}\) = − 43.7° (c = 0.52, in CHCl3), 6D: \(\left[ \alpha \right]_{D}^{25}\) = − 26.6° (c = 0.56, in CHCl3), 1D: m.p. 181–183 °C (literature: 183–185 °C [10], 185–188 °C [12]); \(\left[ \alpha \right]_{D}^{25}\) = − 64.8° (c = 0.43, in CHCl3).
Enzymatic hydrolysis of d- and l-coniferin (1D and 1L) by β-glucosidase
An aqueous solution of β-glucosidase (6 × 10−5 g/L, 5 mL) was added to an aqueous solution of coniferin (0.3 mmol/L, 10 mL). The reaction solution was stirred at 40 °C. An aliquot of the reaction solution (1 mL) was taken out at the prescribed time and poured into distilled water (3 mL) and then 0.1 mL of a 2.5 mM NaOH aqueous solution was immediately added. The mixed solution was subjected to UV–Vis measurement. The amount of coniferyl alcohol formed was determined using a calibration curve of coniferyl alcohol at 311 nm.
Transport assay of d- and l-coniferin (1D and 1L)
The preparation of microsomal fractions from differentiating xylem of poplar (Populus sieboldii × P. grandidentata) and the transport assay were carried out according to the methods used in previous studies [3, 13]. Uptake of coniferin by membrane vesicles was measured at 28 °C for 20 min in 100 μL of reaction mixture [50 mM HEPES–KOH (pH 7.5), 5 mM Mg/ATP, 50 μM substrate and membrane vesicles (ca. 10 μg protein)], unless otherwise stated. Data are reported as technical replicates.