Article pubs.acs.org/JAFC
Synthesis of New Sulfated and Glucuronated Metabolites of Dietary Phenolic Compounds Identified in Human Biological Samples A. Filipa Almeida,†,‡ Cláudia N. Santos,†,‡ and M. Rita Ventura*,‡ †
Instituto de Biologia Experimental e Tecnológica, Universidade Nova de Lisboa Instituto de Tecnologia Quimica e Biologica, Apartado 12, 2781-901 Oeiras, Portugal ‡ Instituto de Tecnologia Quı ́mica e Biológica António Xavier, NOVAUniversidade Nova de Lisboa, Av. da República, 2781-901 Oeiras, Portugal S Supporting Information *
ABSTRACT: (Poly)phenols are a large group of dietary compounds present in fruits and vegetables; their consumption is associated with health beneficial effects. After ingestion, (poly)phenols suffer extensive metabolization, and the identification of their metabolites is an emerging area, because these metabolites are considered the effective bioactive molecules in the human organism. However, a lack of commercially available standards has hampered the study of metabolite bioactivity and the exact structural confirmation in biological samples. New (poly)phenol metabolites previously identified in human samples after the intake of berry juice were chemically synthesized. Efficient chemical reactions were performed with moderate to excellent yields and selectivities. These new compounds could be used as standard chemicals for confirmation of the structure of metabolites in biological samples and will also allow mechanistic studies in cellular models. KEYWORDS: polyphenols, organic synthesis, metabolites, sulfation, glycosylation
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INTRODUCTION Polyphenols are part of a large family of compounds that have a wide structural diversity and differ in their chemical behavior and physicochemical properties. This family of compounds is chemically characterized as molecules with a phenolic moiety, presenting a high structural diversity.1 Polyphenols are abundant in our diet, and their potential health-promoting effects in human cell cultures have been demonstrated in experimental animal and human clinical studies.2 They have shown the ability to limit the development of cancers, cardiovascular diseases, neurodegenerative diseases, diabetes, and osteoporosis.3 However, these effects of polyphenols depend on the amount consumed and their bioavailability,4 which is generally low because the absorbed forms are not the native compounds found in food; they are more likely to be metabolites.5 After the intake and digestion of the parent compounds in the intestine, the aglycones undergo some degree of phase II metabolism, forming sulfate esters, Oglucuronates, and/or O-methyl ether metabolites.6 The consumption of plant-derived beverages and foods containing these polyphenolic compounds may have beneficial effects on human health, as shown in numerous studies. However, structure−activity relationship studies to elucidate their possible modes of action have been hampered by difficulties in isolating these compounds in their pure and structurally defined forms, and in sufficient amounts, from natural sources.7 However, many authors have identified new phenolic metabolites in nutritional intervention studies after the ingestion of fruits and/or vegetables.4,8−11 In a previous work, our group has identified several phenolic metabolites in human plasma and urine, derived from colonic metabolism following the ingestion of a mixed berry fruit purée.9,11 However, for some new metabolites, with predicted © XXXX American Chemical Society
identity by exact mass, it was not possible to confirm their identity due to the lack of standards. Several of these compounds have the same molecular weight, and some have the possibility of presenting isomers, which also have the same molecular weight; without pure standards with a wellcharacterized structure, it is very difficult to identify these compounds from biological samples. The bacterial arylsulfotransferase (AST) from Desulfitobacterium hafniense has been expressed and purified,12 and it was used as a catalytic tool to obtain sulfated natural flavonoid compounds, in the range from 67 nmol to 1.3 mmol.13 Chemical synsthesis has successfully provided polyphenols in pure form and in resonable quantities. Furthermore, organic chemists can also play an important role in the synthesis of new analogues of these natural metabolites, with improved biological properties for different health applications. The synthesis of (poly)phenol metabolites is an emerging and very important area of research. Therefore, the aim of our work is to synthesize new polyphenol metabolites on the basis of data obtained from exact mass identification in human samples, to be used as standards. Efficient chemical reactions were performed using already described sulfation and glucuronidation protocols. These syntheses were successful, with moderate to high yields and selectivities. These new (poly)phenol metabolites will allow the final confirmation of the structure Special Issue: XXVIIIth International Conference on Polyphenols 2016 Received: Revised: Accepted: Published: A
December 16, 2016 February 13, 2017 February 15, 2017 February 15, 2017 DOI: 10.1021/acs.jafc.6b05629 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
CD3OD, and CDCl3 with chemical shift values (δ) in parts per million, and 13C NMR spectra were obtained at 100.61 MHz in the same deuterated solvents. Assignments were supported by 2D correlation NMR studies. Specific rotations ([α]D20) were measured using an automatic polarimeter. Compounds 2a−2j were lyophilized and stored at 2−8 °C. Sulfated derivatives were synthesized using the method described in Figure 3. General Procedure of Sulfation. The phenol (500 mg) and sulfur trioxide−pyridine (1 equiv) were dissolved in 10 mL of anhydrous pyridine and kept at 65 °C with constant stirring for 24 h. The reaction was quenched by the addition of water. Solvents were removed in a vacuum, and the residue was dissolved in water. The unreacted starting materials were separated with ethyl acetate, and the product was purified on a Dowex 50W-X8 ion-exchange resin (SigmaAldrich) loaded with Na+. The column was eluted with water. The final residue was dried under vacuum and characterized by 1H and 13C NMR. Guaiacol-O-sulfate (1a): 644 mg, 78% yield; 1H NMR (400 MHz, D2O) δ 7.27 (dd, J = 1.28 Hz, J = 8.09 Hz, 1H, Harom), 7.19 (td, J = 1.45 Hz, J = 8.32 Hz, J = 8.84 Hz, 1H, Harom), 7.05 (d, J = 7.45 Hz, 1H, Harom), 6.93 (td, J = 1.16 Hz, J = 8.03 Hz, J = 8.84 Hz, 1H, Harom), 3.77 (s, 3H, Me); 13C NMR (100 MHz, D2O) δ 155.2 (Cqarom), 139.8 (Cqarom), 127.3 (Carom), 122.8 (Cqarom), 121.1 (Carom), 113.5 (Carom), 55.9 (CH3). Benzoic acid 4-O-sulfate (1b): 473 mg, 68% yield; 1H NMR (400 MHz, D2O) δ 7.98 (d, J = 8.95 Hz, 2H, Harom), 7.33 (d, J = 8.95 Hz, 2H, Harom); 13C NMR (100 MHz, D2O) δ 170.0 (CO), 155.1 (Cqarom), 131.6 (Carom), 127.4 (Cqarom), 121.1 (Carom). Protocatechuic acid 4-O-sulfate (1c) and protocatechuic acid 3O-sulfate (1d): 440 mg, 58% yield, mixture of both compounds in a proportion 1:2.03; 1H NMR (400 MHz, D2O) δ 7.87 (d, J = 2.14 Hz, 1H, Harom(3)), 7.72 (dd, J = 8.54 Hz, J = 2.47 Hz, 1H, Harom(3)), 7.48 (d, J2,6 = 2.07 Hz, 1H, Harom(4)), 7.44 (dd, J = 2.34 Hz, J = 8.38 Hz, 1H, Harom(4)), 7.36 (d, J = 8.51 Hz, 1H, Harom(4)), 6.99 (d, J = 8.51 Hz, 1H, Harom(3)); 13C NMR (100 MHz, D2O) δ 170.8 (CO), 152.7 (Cqarom), 138.3 (Cqarom), 129.0 (Carom(3)), 124.5 (Cqarom(3)), 123.5 (Cqarom), 122.4 (Carom(4)), 121.9 (Carom(4)), 118.1 (Carom(4)), 117.0 (Carom(3)). Isovanillic acid 3-O-sulfate (1e): 443 mg, 60% yield; 1H NMR (400 MHz, D2O) δ 7.86 (d, J = 1.89 Hz, 1H, Harom), 7.83 (dd, J = 1.72 Hz, J
of the metabolites found in human plasma samples, and further work will be developed using them for mechanistic studies in cell-based assays. This paper shows the methodologies used in the synthesis of phenolic sulfates 1a−1g (Figure 1) and glucuronides 2a−2j (Figure 2), which were identified in previous human studies.9,11
Figure 1. Structures of sulfate derivatives.
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MATERIALS AND METHODS
Reagents and solvents were purified and dried as described.14 All reactions were carried out under an inert atmosphere (argon) except when the solvents were not dried. The reactions’ progress was monitored by TLC on aluminum-backed silica gel (Merck 60 F254). Flash preparative column chromatography was performed with Silica Gel Merck 60. 1H NMR spectra were obtained at 400 MHz in D2O,
Figure 2. Structures of glucuronate derivatives. B
DOI: 10.1021/acs.jafc.6b05629 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 3. Synthesis of sulfate derivatives. Reagents and conditions: (i) pyridine, 65 °C, 24 h. The yields are of isolated products. = 8.53 Hz, 1H, Harom), 7.12 (d, J = 9.02 Hz,1H, Harom), 3.86 (s, 3H, Me); 13C NMR (100 MHz, D2O) δ 155.3 (Cqarom), 139.3 (Cqarom), 129.1 (Carom), 124.0 (Carom), 112.7 (Carom), 56.1 (CH3). Caffeic acid 4-O-sulfate (1f) and caffeic acid 3-O-sulfate (1g): 498 mg, 69% yield, mixture of both compounds in approximately equal amounts (1:1.27) with a contamination of disulfate; 1H NMR (400 MHz, D2O) δ 7.57−7.52 (m, 3H, Harom), 7.35 (dd, J = 2.07 Hz, J = 8.48 Hz, 1H, Harom(3)), 7.31 (d, J = 8.40 Hz, 1H, CH(4)), 7.17 (d, J = 2.00 Hz, 1H, Harom(4)), 7.12 (dd, J = 2.09 Hz, J = 8.43 Hz, 1H, Harom(4)), 6.96 (d, J = 8.44 Hz, 1H, CH(3)), 6.37 (d, J = 16.11 Hz, 1H, CH(4)), 6.31 (d, J = 16.11 Hz, 1H, CH(3)); 13C NMR (100 MHz, D2O) δ 171.2 (CO(3)), 171.0 (CO(4)), 150.5 (Cqarom), 148.2 (Cqarom), 145.5 (CHCH), 145.1 (CHCH), 140.6 (Cqarom), 139.0 (Cqarom), 133.0 (Cqarom), 128.6 (Carom), 127.9 (Carom), 126.9 (Cqarom), 124.0, 123.0, 122.6, 121.1, 117.9, 117.6, 116.9, 116.4, 115.5 (Carom). Preparation of Glucuronates and Precursors. The synthesis of glucuronate derivatives involved three stepsfirst, the glucuronidation with different phenols and the glucuronic acid donor. Removal of the acetyl groups followed by the hydrolysis of the esters afforded the final compounds, identified in biological samples. General Procedure of Glucuronidation. The phenol (0.7 equiv) and methyl 2,3,4-tri-O-acetyl-1-O-trichloroacetimidoyl-α-D-glucuronate (0.250g, 0.5 mmol) were dissolved in anhydrous dichloromethane (19 mL) in the presence of 4 Å molecular sieves. The reaction was stirred at room temperature for 30 min and then cooled to −20 °C, and BF3·OEt2 (0.2 equiv) was added dropwise. The mixture was allowed to warm gradually to room temperature under continued stirring for 4 h. The reaction was quenched by adding saturated NaHCO3 solution. The organic layer was extracted with dichloromethane and dried over anhydrous Na2SO4. The residue was purified by column chromatography that was eluted with hexane/AcOEt (2:1) to give the glucuronide conjugate. Methyl 2,3,4-tri-O-acetyl-1-O-(2-hydroxyphenyl)-β-D-glucuronate (6a): yellow solid, 94 mg, 48% yield; 1H NMR (400 MHz, CDCl3) δ 7.03 (t, J = 7.98 Hz, 1H, Harom), 6.98−6.95 (m, 2H, Harom), 6.83 (t, J = 7.91 Hz, 1H, Harom), 5.40−5.26 (m, 3H, H-2, H-3 and H-4), 5.02 (d, J1,2 = 7.8 Hz, 1H, H-1), 4.16 (d, J4,5 = 8.8 Hz, 1H, H-5), 3.77 (s, 3H, OCH3), 2.12 (s, 3H, CH3−OAc), 2.06 (s, 3H, CH3-OAc), 2.05 (s, 3H, CH3-OAc); 13C NMR (100 MHz, CDCl3) δ 169.9 (CO), 169.8 (CO), 169.4 (CO), 166.7 (COsugar), 147.5 (Cqarom), 144.1 (Cqarom), 125.5 (Carom), 120.4 (Carom), 117.9 (Carom), 116.6 (Carom), 101.5 (C-1), 72.6 (C-5), 71.4, 71.1, 69.0 (C-2, C-3, C-4), 53.2 (OCH3), 20.7 (CH3−OAc), 20.6 (CH3−OAc), 20.5 (CH3-OAc). Other characterization data were described in ref 15. Methyl 2,3,4-tri-O-acetyl-1-O-(1,3-dihydroxyphenyl)-β-D-glucuronate (6b): white solid, 80 mg, 17% yield; [α]D20 −33.14 (c 0.7, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 6.95 (t, J = 8.34 Hz, 1H, Harom), 6.52 (d, J = 8.27 Hz, 2H, Harom), 5.33−5.28 (m, 3H, H-2, H-3 and H-4), 4.91 (d, J1,2 = 7.40 Hz, 1H, H-1), 4.14 (d, J4,5 = 9.26 Hz, 1H, H-5), 3.79 (s, 3H, OCH3), 2.16 (s, 3H, CH3−OAc), 2.06 (s, 3H, CH3−OAc), 2.05 (s, 3H, CH3−OAc); 13C NMR (100 MHz, CDCl3) δ 170.0 (CO), 169.4 (CO), 169.2 (CO), 166.3 (COsugar), 149.3 (Cqarom), 132.2 (Cqarom), 126.8 (Carom), 108.6 (Carom), 103.0 (C-
1), 72.3 (C-5), 71.7, 71.4, 68.5 (C-2, C-3, C-4), 53.3 (OCH3), 20.6 (CH3−OAc), 20.6 (CH3−OAc), 20.5 (CH3−OAc). Methyl 2,3,4-tri-O-acetyl-1-O-(2-methoxyphenyl)-β-D-glucuronate (6c): white solid, 248 mg, 54% yield; [α]D20 −46.0 (c 1.0, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.15 (dd, J = 7.19 Hz, J = 0.82 Hz, 1H, Harom), 7.08 (t, J = 8.28 Hz, 1H, Harom), 6.91−6.86 (m, 2H, Harom), 5.34−5.30 (m, 3H, H-2, H-3, and H-4), 5.03 (d, J1,2 = 7.17 Hz, 1H, H-1), 4.09 (d, J4,5 = 9.02 Hz, 1H, H-5), 3.82 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 2.08 (s, 3H, CH3−OAc), 2.05 (s, 3H, CH3− OAc), 2.03 (s, 3H, CH3−OAc); 13C NMR (100 MHz, CDCl3) δ 170.2 (CO), 169.4 (CO), 169.3 (CO), 167.0 (COsugar), 150.8 (Cqarom), 145.8 (Cqarom), 125.0 (Carom), 120.9 (Carom), 120.8 (Carom), 112.7 (Carom), 100.8 (C-1), 72.7 (C-5), 71.9, 71.1, 69.4 (C-2, C-3, C4), 56.0 (OCH3), 52.9 (OCH3), 20.6 (CH3−OAc), 20.5 (CH3−OAc). Methyl 2,3,4-tri-O-acetyl-1-O-(1-hydroxy-4-methylphenyl)-β-Dglucuronate (6d) and methyl 2,3,4-tri-O-acetyl-1-O-(2-hydroxy-4methylphenyl)-β-D-glucuronate (6e): mixture of both compounds in a proportion of 1:1.42; white solid, 177 mg, 38% yield; 1H NMR (400 MHz, CDCl3) δ 6.85−6.83 (m, 3H, Harom), 6.78−6.76 (m, 2H, Harom), 6.62 (dd, J = 1.53 Hz, J = 8.04 Hz, 1H, Harom), 5.37−5.25 (m, 6H, H2(4d), H-3(4d), H-4(4d), H-2(4e), H-3(4e) and H-4(4e)), 5.00 (d, J1,2 = 7.42 Hz, 1H, H-1(4d)), 4.96 (d, J1,2 = 7.33 Hz, 1H, H-1(4e)), 4.17−7.12 (m, 2H, H-5(4d) and H-5(4e)), 3.77 (s, 6H, OCH3(4d) and OCH3(4e)), 2.27 (s, 6H, CH3−OAc), 2.11 (s, 6H, CH3), 2.06 (s, 6H, CH3−OAc), 2.05 (s, 6H, CH3−OAc); 13C NMR (100 MHz, CDCl3) δ 169.9(6) (C O), 169.9(5) (CO), 169.7(4) (CO), 169.6(9) (CO), 169.4 (CO), 166.7(2) (COsugar), 166.6(9) (COsugar), 147.3 (Cqarom), 145.2 (Cqarom), 143.7 (Cqarom), 141.9 (Cqarom), 135.7 (Cqarom), 130.0 (Cqarom), 125.9 (Carom), 120.8 (Carom), 118.5 (Carom), 118.2 (Carom), 117.2 (Carom), 116.2 (Carom), 101.9 (C-1(4e)), 101.6 (C-1(4d)), 72.6 (C5(4e)), 72.5 (C-5(4d)), 71.5, 71.4, 71.1, 69.0(4), 69.0(2) (C-2(4e), C3(4e), C-4(4e), C-2(4d), C-3(4d), C-4(4d)), 53.1 (OCH3), 21.0 (CH3− OAc), 20.7 (CH3−OAc), 20.6 (CH3−OAc), 20.5 (CH3−OAc). Methyl 2,3,4-tri-O-acetyl-1-O-(4-benzylcarbonylphenyl)-β-D-glucuronate (6f): white solid, 204 mg, 36% yield; [α]D20 −21.67 (c 0.6, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 8.04 (d, 2H, Harom), 7.45− 7.34 (m, 5H, CH(Bn)), 7.01 (d, J = 8.87 Hz, 2H, Harom), 5.38−5.30 (m, 5H, CH2(Bn), H-2, H-3, and H-4), 5.24 (d, J1,2 = 7.04 Hz, 1H, H-1), 4.22 (d, J4,5 = 8.69 Hz, 1H, H-5), 3.71 (s, 3H, OCH3), 2.06 (s, 3H, CH3−OAc), 2.05 (s, 3H, CH3−OAc); 13C NMR (100 MHz, CDCl3) δ 170.1 (CO), 169.2 (CO), 166.7 (CO), 165.7 (CO), 160.1 (CO), 131.8 (Carom), 131.6 (Cqarom), 128.6 (Carom), 128.4 (Carom), 128.1 (Carom), 127.9 (Cqarom), 125.3 (Cqarom), 116.3 (Carom), 98.3 (C1), 72.7 (C-5), 71.7, 71.0, 68.9 (C-2, C-3, and C-4), 66.7 (CH2), 53.0 (OCH3), 20.6(1) (CH3−OAc), 20.6(0) (CH3−OAc), 20.5 (CH3− OAc). Methyl 2,3,4-tri-O-acetyl-1-O-(2-hydroxy-4-benzylcarbonylphenyl)-β-D-glucuronate (6g) and methyl 2,3,4-tri-O-acetyl-1-O-(2hydroxy-5-benzylcarbonylphenyl)-β-D-glucuronate (6h): mixture of both compounds in a proportion of 1:1.27; white solid, 114 mg, 24% yield; 1H NMR (400 MHz, CDCl3) δ 7.81 (dd, J = 1.71 Hz, J = 8.46 Hz, 1H, Harom), 7.67 (dd, 2H, Harom), 7.61 (d, J = 8.48 Hz, 1H, Harom), 7.43−7.32 (m, 10H, CH(Bn)), 6.99 (t, 2H, Harom), 5.38−5.27 (m, 10H, C
DOI: 10.1021/acs.jafc.6b05629 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
3.55 (m, 3H, H-2, H-3, and H-4); 13C NMR (100 MHz, D2O) δ 170.5 (CO), 148.8 (Cqarom), 145.1 (Cqarom), 124.2 (Carom), 121.4 (Carom), 116.6 (Carom), 113.0 (Carom), 100.6 (C-1), 74.9 (C-3), 74.5 (C-5), 72.5 (C-2), 71.0 (C-4), 55.6 (OCH3), 53.0 (OCH3). Methyl 1-O-(1-hydroxy-4-methylphenyl)-β-D-glucuronate (7d) and methyl 1-O-(2-hydroxy-4-methylphenyl)-β-D-glucuronate (7e): mixture of both compounds in a proportion of 1:1.42; white solid, 100 mg, 91% yield; 1H NMR (400 MHz, CD3OD) δ 6.97 (d, J = 8.53 Hz, 1H, Harom(7e)), 6.93 (s, 1H, Harom(7d)), 6.75 (s, 2H, Harom(7d)), 6.69 (d, J = 1.79 Hz, 1H, Harom(7e)), 6.60 (dd, 1H, Harom(7e)), 4.82 (d, J1,2 = 7.16 Hz, 1H, H-1(7d)), 4.77 (d, J1,2 = 7.16 Hz, 1H, H-1(7e)), 4.00 (t, 2H, H5), 3.82 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.64 (td, 2H, H-4(7e) and H-4(7d)), 3.56−3.47 (m, 4H, H-2(7e), H-3(7e), H-2(7d), and H-3(7d)), 2.24 (S, 6H, OCH3); 13C NMR (100 MHz, CD3OD) δ 177.1 (C O), 176.7 (CO), 133.8 (Cqarom), 129.2 (Cqarom), 124.1 (Carom), 119.9 (Carom), 118.3 (Carom), 117.7 (Carom), 116.5(Carom), 115.6 (Carom), 103.5 (C-1(7e)), 103.2 (C-1(7d)), 75.4(3) (C-3), 75.3(8) (C-5), 73.2 (C-2), 71.6 (C-4), 51.5 (OCH3), 19.5 (CH3(7e)), 19.4 (CH3(7d)). Methyl 1-O-(4-benzoxycarbonylphenyl)-β-D-glucuronate (7f): white solid, 100 mg, 100% yield; [α]D20 −77.50 (c 0.2, MeOH); 1H NMR (400 MHz, CD3OD) δ 8.02 (d, J = 8.63 Hz, 2H, Harom), 7.47− 7.35 (m, 5H, CH(Bn)), 7.15 (d, J = 8.63 Hz, 2H, Harom), 5.35 (s, 2H, CH2(Bn)), 5.14 (d, J1,2 = 7.79 Hz, 1H, H-1), 4.12 (d, J4,5 = 9.36 Hz, 1H, H-5), 3.79 (s, 3H, OCH3), 3.67−3,62 (m, 1H, H-4), 3.56−3.52 (m, 2H, H-2 and H-3); 13C NMR (100 MHz, CDCl3) δ 169.4 (CO), 166.0 (CO), 161.2 (Cqarom), 136.3 (Cqarom), 131.1 (Carom), 131.0 (Carom), 128.2 (Carom), 127.8 (Carom), 127.7 (Carom), 124.0 (Cqarom), 115.8(1) (Carom), 115.7(6) (Carom), 100.7 (C-1), 75.6 (C-3), 75.3 (C5), 73.0 (C-2), 71.4 (C-4), 66.2 (CH2), 51.5 (OCH3). Methyl 1-O-(2-hydroxy-4-benzoxycarbonylphenyl)-β-D-glucuronate (7g) and methyl 1-O-(2-hydroxy-5-benzoxycarbonylphenyl)β-D-glucuronate (7h): white solid, 59 mg, 70% yield; mixture of both compounds in a proportion of 1:1.27; 1H NMR (400 MHz, CD3OD) δ 7.81 (d, J = 2.06 Hz, 1H, Harom(7h)), 7.71 (dd, J = 8.43 Hz, 1H, Harom(7h)), 7.56−7.53 (m, 2H, Harom(7g)), 7.47−7.33 (m, 10H, CH(Bn)), 7.16 (d, J = 8.20 Hz, 1H, Harom(7g)), 6.93 (d, 1H, Harom(7h)), 5.34 (s, 4H, CH2(Bn)(7g) and CH2(Bn)(7h)), 5.02 (d, J1,2 = 7.49 Hz, 1H, H-1(7g)), 4.93 (d, J1,2 = 7.33 Hz, 1H, H-1(7h)), 4.10 (d, J4,5 = 9.86 Hz, 1H, H5(7g)), 4.05 (d, J4,5 = 9.86 Hz, 1H, H-5(7h)), 3.80 (s, 3H, OCH3(7g)), 3.76 (s, 3H, OCH3(7h)), 3.68−3.63 (m, 2H, H-4(7h) and H-4(7g)), 3.61−3.50 (m, 4H, H-2(7h), H-3(7h), H-2(7g), and H-3(7g));13C NMR (100 MHz, CD3OD) δ 169.4 (CO), 166.1 (CO), 152.3 (Cqarom), 128.2 (Carom(7h)), 128.2 (Carom(7g)), 127.8(0) (Carom), 127.7(6) (Carom), 127.7 (Carom), 127.6 (Carom), 126.1 (Carom), 121.7 (Carom), 121.4 (Cqarom), 118.7 (Carom(7h)), 116.8(Carom(7g)), 115.8 (Carom(7h)), 115.6 (Carom(7g)), 102.7 (C-1(7h)), 101.5 (C-1(7g)), 75.3(5) (C-3), 75.3(4) (C-5), 73.1, 73.0, 71.5(3), 71.4(9) (C-3(7g), C-4(7g), C-3(7h) and C4(7h)), 66.2 (CH2(7g)), 66.1 (CH2(7h)), 51.5 (OCH3). Methyl 1-O-(2-methoxy-4-benzoxycarbonylphenyl)-β-D-glucuronate (7i): white solid, 127 mg, 76% yield; 1H NMR (400 MHz, CD3OD) δ 7.69−7.63 (m, 2H, Harom), 7.48−7.35 (m, 5H, CH(Bn)), 7.17 (d, J = 8.43 Hz, 1H, Harom), 5.36 (s, 2H, CH2(Bn)), 5.14 (d, J1,2 = 7.51 Hz, 1H, H-1), 4.08 (d, J4,5 = 10.09 Hz, 1H, H-5), 3.91 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.65 (t, J3,4 = 9.23 Hz, 1H, H-4), 3.60− 3.50 (m, 2H, H-2 and H-3); 13C NMR (100 MHz, CD3OD) δ 169.5 (CO), 149.8 (Cqarom), 146.2 (Cqarom), 123.2 (Carom), 120.7 (Carom), 117.2 (Carom), 112.5 (Carom), 101.6 (C-1), 75.6 (C-3), 75.3 (C-5), 73.2 (C-2), 71.6 (C-4), 52.2 (OCH3), 51.4 (OCH3). Methyl 1-O-(2-methoxy-5-benzoxycarbonylphenyl)-β-D-glucuronate (7j): white solid, 102 mg, 65% yield; 1H NMR (400 MHz, CD3OD) δ 7.81 (dd, J = 2.07 Hz, J = 8.59 Hz, 1H, Harom), 7.77 (d, J = 2.10 Hz, 1H, Harom), 7.47−7.35 (m, 5H, CH(Bn)), 7.11 (d, J = 8.24 Hz, 1H, Harom), 5.33 (d, 2H, CH2(Bn)), 5.03 (d, J1,2 = 7.42 Hz, 1H, H-1), 4.01 (d, J4,5 = 9.61 Hz, 1H, H-5), 3.94 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.65 (t, J = 8.68 Hz, 1H, H-4), 3.59−3.48 (m, 2H, H-2 and H3); 13C NMR (100 MHz, CD3OD) δ 169.4 (CO), 166.0 (CO), 154.2 (Cqarom), 145.7 (Cqarom), 128.2 (Carom), 127.8 (Carom), 127.6 (Carom), 125.6 (Carom), 122.3 (Cqarom), 118.0 (Carom), 111.5 (Carom), 101.4 (C-1), 75.6 (C-3), 75.3 (C-5), 73.2 (C-4), 71.5 (C-2), 66.2 (CH2), 55.2 (OCH3), 51.5 (OCH3).
H-2(6g), H-3(6g), H-4(6g), CH2(Bn)(6g), H-2(6h), H-3(6h), H-4(6h) and CH2(Bn)(6h)), 5.11 (d, J = 7.25 Hz, 1H, H-1(6g)), 5.07 (d, J = 7.25 Hz, 1H, H-1(6h)), 4.20 (t, 2H, H-5(6g) and H-5(6h)), 3.76 (s, 3H, OCH3(6g)), 3.75 (s, 3H, OCH3(6h)), 2.10 (s, 3H, CH3−OAc), 2.09 (s, 3H, CH3− OAc), 2.06 (s, 3H, CH3−OAc); 13C NMR (100 MHz, CDCl3) δ 169.9 (CO), 169.4 (CO), 152.1(Cqarom), 128.6 (Carom), 128.2 (Carom), 128.1 (Carom), 127.8 (Carom), 119.5 (Cqarom), 116.3 (Carom), 116.1 (Carom), 101.4 (C-1), 72.7 (C-5(6g)), 72.6 (C-5(6h)), 71.2, 71.0, 68.9 (C2, C-3 and C-4), 66.7 (CH2(6g)), 66.7 (CH2(6h)), 53.2 (OCH3), 20.7 (CH3−OAc), 20.6 (CH3−OAc), 20.5 (CH3−OAc). Methyl 2,3,4-tri-O-acetyl-1-O-(2-methoxy-4-benzylcarbonylphenyl)-β-D-glucuronate (6i): white solid, 220 mg, 37% yield; [α]D20 −32.67 (c 0.3, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.66 (dd, J = 1.88 Hz, J = 8.44 Hz, 1H, Harom), 7.61 (d, J = 1.95 Hz, 1H, Harom), 7.44−7.34 (m, 5H, CH(Bn)), 7.14 (d, J = 8.44 Hz, 1H, Harom), 5.35−5.29 (m, 5H, H-2, H-3, H-4, CH2(Bn)), 5.13 (d, J = 6.74 Hz, 1H, H-1), 4.13 (d, J = 8.15 Hz, 1H, H-5), 3.86 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 2.07 (s, 3H, CH3−OAc), 2.05 (s, 3H, CH3− OAc), 2.04 (s, 3H, CH3−OAc); 13C NMR (100 MHz, CDCl3) δ 170.1 (CO), 169.3 (CO), 166.8 (CO), 149.6 (Cqarom), 128.6 (Carom), 128.3 (Carom), 128.1 (Carom), 126.4 (Cqarom), 123.2 (Carom), 119.0 (Carom), 113.7 (Carom), 100.0 (C-1), 72.7 (C-5), 71.7, 71.0, 69.1 (C-2, C-3, and C-4), 66.8 (CH2), 56.2 (OCH3), 52.7 (OCH3), 20.6(4) (CH3−OAc), 20.6(1) (CH3−OAc), 20.5 (CH3−OAc). Methyl 2,3,4-tri-O-acetyl-1-O-(2-methoxy-5-benzylcarbonylphenyl)-β-D-glucuronate (6j): white solid, 217 mg, 48% yield; [α]D20 +1.0 (c 0.3, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.86 (dd, J = 1.90 Hz, J = 8.55 Hz, 1H, Harom), 7.83 (d, J = 1.90 Hz, 1H, Harom), 7.44−7.32 (m, 5H, CH(Bn)), 6.91 (d, J = 9.09 Hz, 1H, Harom), 5.36−5.30 (m, 5H, H-2, H-3, H-4, CH2(Bn)), 5.09 (d, J = 7.06 Hz, 1H, H-1), 4.12 (d, J = 9.09 Hz, 1H, H-5), 3.88 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 2.07 (s, 3H, CH3−OAc), 2.05 (s, 3H, CH3−OAc), 2.04 (s, 3H, CH3−OAc); 13C NMR (100 MHz, CDCl3) δ 170.1 (CO), 169.4 (CO), 169.2 (CO), 166.8 (CO), 165.6 (CO), 154.7 (Cqarom), 145.2 (Cqarom), 136.1 (Cqarom), 128.6 (Carom), 128.2 (Carom), 128.1 (Carom), 127.3 (Carom), 124.7 (Cqarom), 121.3 (Carom), 111.6 (Carom), 100.4 (C-1), 72.6 (C-5), 71.8, 71.1, 69.1 (C-2, C-3 and C-4), 66.6 (CH2), 56.1 (OCH3), 52.9 (OCH3), 20.6(4) (CH3−OAc), 20.6(1) (CH3−OAc), 20.5 (CH3−OAc). General Procedure of Deacetylation. A solution of sodium methoxide in methanol (1M) (0.6 equiv) was added to a solution of the glucuronate in methanol (1.5 mL per 100 mg), and the mixture was stirred at room temperature for 15 min. Then, previously activated Dowex-H+ resin was added until the pH of the reaction mixture reached 7. After filtration with methanol, the solvent was removed under vacuum, and the crude was purified by preparative chromatography eluted with AcOEt, affording the final product. Methyl 1-O-(2-hydroxyphenyl)-β-D-glucuronate (7a): yellow solid, 55 mg, 98% yield; 1H NMR (400 MHz, CD3OD) δ 7.10 (dd, J = 1.41 Hz, J = 7.93 Hz, 1H, Harom), 6.94 (td, J = 9.19 Hz, 1H, Harom), 6.86 (dd, 1H, Harom), 6.79 (td, 1H, Harom), 4.88 (s, 1H, H-1), 4.02 (d, J4,5 = 9.60 Hz, 1H, H-5), 3.81 (s, 3H, OCH3), 3.65 (t, J3,4 = 8.99 Hz, 1H, H4), 3.58−3.48 (m, 2H, H-2 and H-3); 13C NMR (100 MHz, CD3OD) δ 169.5 (CO), 147.2 (Cqarom), 145.1 (Cqarom), 123.8 (Carom), 119.6 (Carom), 117.6 (Carom), 115.9 (Carom), 103.0 (C-1), 75.3(9) (C-3), 75.3(5) (C-5), 73.1 (C-2), 71.6 (C-4), 51.6 (OCH3). Other characterization data were described in ref15. Methyl 1-O-(1,3-dihydroxyphenyl)-β-D-glucuronate (7b): white solid, 62 mg, 96% yield; 1H NMR (400 MHz, CD3OD) δ 6.73 (t, J = 8.23 Hz, 1H, Harom), 6.27 (d, 2H, Harom), 4.59 (d, J1,2 = 7.15 Hz, 1H, H-1), 3.85 (d, J4,5 = 9.06 Hz, 1H, H-5), 3.69 (s, 3H, OCH3), 3.50− 3.35 (m, 3H, H-2, H-3, and H-4); 13C NMR (100 MHz, CD3OD) δ 169.7 (CO), 150.1 (Cqarom), 133.1 (Cqarom), 125.4 (Carom), 107.4 (Carom), 106.1 (C-1), 75.6 (C-5), 75.5, 73.4, 71.5 (C-2, C-3, C-4), 51.6 (OCH3). Methyl 1-O-(2-methoxyphenyl)-β-D-glucuronate (7c): white solid, 33 mg, 46% yield; [α]D20 −81.17 (c 0.6, CH3OH); 1H NMR (400 MHz, D2O) δ 7.06−6.98 (m, 3H, Harom), 6.87 (td, J = 1.60 Hz, J = 7.82 Hz, 1H, Harom), 5.05 (d, J1,2 = 7.30 Hz, 1H, H-1), 4.09 (d, J4,5 = 9.25 Hz, 1H, H-5), 3.76 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.63− D
DOI: 10.1021/acs.jafc.6b05629 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 4. Synthesis of glucuronate derivatives. Reagents and conditions: (i) BF3·OEt2, DCM, −20 °C to room temperature, 4 h; (ii) MeONa, MeOH, room temperature, 30 min; (iii) LiOH:H2O, room temperature, 5 min. The yields are of isolated products. 4H, OCH3 and H-5), 3.59−3.50 (m, 3H, H-2, H-3, and H-4); 13C NMR (100 MHz, D2O) δ 175.4 (CO), 148.6 (Cqarom), 145.3 (Cqarom), 123.8 (Carom), 121.5 (Carom), 116.3 (Carom), 112.9 (Carom), 100.3 (C-1), 76.3 (C-3), 75.4 (C-5), 72.7 (C-2), 71.7 (C-4), 55.8 (OCH3). 1-O-(1-Hydroxy-4-methylphenyl)-β-D-glucuronic acid (2d) and 1O-(2-hydroxy-4-methylphenyl)-β-D-glucuronic acid (2e): white solid, 62 mg, 73% yield; 1H NMR (400 MHz, CD3OD) δ 7.13 (d, J = 8.05 Hz, 1H, Harom(2e)), 7.08 (s, 1H, Harom(2d)), 6.73 (s, 2H, Harom(2d)), 6.68 (d, J = 1.53 Hz, 1H, Harom(2e)), 6.59 (dd, 1H, Harom(2e)), 4.72 (d, J1,2 = 7.07 Hz, 1H, H-1(2d)), 4.68 (d, J1,2 = 7.15 Hz, 1H, H-1(2e)), 3.70 (t, 2H, H-5(2d) and H-5(2e)), 3.56−3.49 (m, 6H, H-2(2e), H-2(2d), H-3(2e), H3(2d), H-4(2e), and H-4(2d)), 2.24 (s, 3H, CH3(2d)), 2.24 (s, 3H, CH3(2e)); 13C NMR (100 MHz, CD3OD) δ 176.8 (CO), 175.0 (CO), 133.7 (Cqarom), 129.2 (Cqarom), 124.1 (Carom), 120.0 (Carom), 119.3 (Carom), 118.7 (Carom), 116.4 (Carom), 115.6 (Carom), 104.0 (C1(2e)), 103.7 (C-1(2d)), 76.1 (C-3), 75.3 (C-5), 73.3 (C-2), 72.2 (C-4), 19.5 (CH3(2e)), 19.3 (CH3(2d)). 1-O-(4-Carboxylphenyl)-β-D-glucuronic acid (2f): white solid, 83 mg, 100% yield; 1H NMR (400 MHz, CD3OD) δ 7.96 (d, J = 8.50 Hz, 2H, Harom), 7.13 (d, J = 8.61 Hz, 2H, Harom), 5.03 (d, J = 7.79 Hz, 1H, H-1), 3.83 (d, J = 9.36 Hz, 1H, H-5), 3.58−3.53 (m, 2H, H-2, H-3, and H-4; 13C NMR (100 MHz, CDCl3) δ 174.9 (CO), 171.3 (C O), 160.4 (Cqarom), 130.9 (Carom), 128.4 (Cqarom), 115.5 (Carom), 100.5 (C-1), 76.3 (C-3), 75.2 (C-5), 73.2 (C-2), 72.1 (C-4). Other characterization data were described in ref 16.
General Procedure for Ester Hydrolysis. A solution of lithium hydroxide (1 M) in water (2 equiv) was added to a solution of compounds 7a−7j in water (1 mL per 100 mg). The mixture was stirred at room temperature during 5 min. Then, the crude mixture was purified by the addition of Dowex-H+ resin previously activated, and the glucuronide was obtained after concentration of solvent. 1-O-(2-hydroxyphenyl)-β-D-glucuronic acid (2a): yellow solid, 48 mg, 100% yield; 1H NMR (400 MHz, CD3OD) δ 7.26 (dd, J = 1.42 Hz, J = 7.89 Hz, 1H, Harom), 6.93 (td, J = 1.47 Hz, J = 7.96 Hz, J = 8.87 Hz, 1H, Harom), 6.85 (dd, J = 1.66 Hz, J = 7.96 Hz, 1H, Harom), 6.78 (td, J = 1.66 Hz, J = 7.96 Hz, J = 9.06 Hz, 1H, Harom), 4.74 (d, 1H, J1,2 = 7.57 Hz, H-1), 3.71 (d, J4,5 = 9.28 Hz, 1H, H-5), 3.58−3.48 (m, 3H, H-2, H-3, and H-4); 13C NMR (100 MHz, CD3OD) δ 175.0 (CO), 147.4 (Cqarom), 145.7 (Cqarom), 123.7 (Carom), 119.6 (Carom), 118.6 (Carom), 115.8 (Carom), 103.6 (C-1), 76.1 (C-3), 75.3 (C-5), 73.3, 72.2 (C-2 and C-4). Other characterization data were described in ref 15. 1-O-(1,3-Dihydroxyphenyl)-β-D-glucuronic acid (2b): yellow solid, 54 mg, 91% yield; [α]D20 −63.27 (c 1.0, CH3OH); 1H NMR (400 MHz, CD3OD) δ 6.84 (t, J = 7.86 Hz, 1H, Harom), 6.36 (d, 2H, Harom), 4.57 (d, J1,2 = 7.46 Hz, 1H, H-1), 3.68 (d, J4,5 = 9.95 Hz, 1H, H-5), 3.58−3.48 (m, 3H, H-2, H-3, and H-4); 13C NMR (100 MHz, CD3OD) δ 174.8 (CO), 150.2 (Cqarom), 125.3 (Carom), 107.4 (Carom), 106.1 (C-1), 76.1 (C-3), 75.3, 73.5, 72.0 (C-2, C-5, and C-4). 1-O-(2-Methoxyphenyl)-β-D-glucuronic acid (2c): white solid, 16 mg, 80% yield; [α]D20 −85.99 (c 0.8, CH3OH); 1H NMR (400 MHz, D2O) δ 7.11 (d, J = 8.03 Hz, 1H, Harom), 7.04 (bs, 2H, Harom), 6.95− 6.93 (m, 1H, Harom), 5.05 (d, J1,2 = 6.80 Hz, 1H, H-1), 3.80−3.72 (m, E
DOI: 10.1021/acs.jafc.6b05629 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry 1-O-(2-Hydroxy-4-carbonylphenyl)-β-D-glucuronic acid (2g) and 1-O-(2-hydroxy-5-carbonylphenyl)-β-D-glucuronic acid (2h): white solid, 34 mg, 76% yield; 1H NMR (400 MHz, D2O) δ 7.60 (d, J = 1.98 Hz, 1H, Harom(2h)), 7.51 (dd, J = 8.30 Hz, J = 1.83 Hz,1H, Harom(2h)), 7.40−7.36 (m, 2H, Harom(2g)), 7.09 (d, J = 8.79 Hz, 1H, Harom(2g)), 6.89 (d, J = 8.09 Hz, 1H, Harom(2h)), 5.08 (d, J = 7.39 Hz, 1H, H-1(2g)), 5.04 (d, J = 7.54 Hz, 1H, H-1(2h)), 3.84 (d, J = 9.42 Hz, 2H, H-5(2g) and H5(2h)), 3.61−3.50 (m, 6H, H-2(2h), H-2(2g), H-3(2h), H-3(2g), H-4(2h), and H-4(2g)); 13C NMR (100 MHz, D2O) δ 175.2 (CO), 172.3 (CO), 171.8 (CO), 149.9 (Cqarom(2h) ), 147.9 (Cq arom(2g) ), 144.8 (Cqarom(2g)), 144.0 (Cqarom(2h)), 128.2 (Cqarom(2g)), 126.1 (Carom(2h)), 124.8 (Cqarom(2h)), 122.6 (Carom(2g)), 117.9 (Carom(2h)), 117.2 (Carom(2g)), 116.0 (Carom(2h)), 115.4 (Carom(2g)), 101.0 (C-1(2h)), 100.2 (C-1(2g)), 76.2 (C-3(2g) and C-3(2h)), 75.1 (C-5(2g) and C-5(2h)), 72.6 (C-2(2g) and C-2(2h)), 71.6 (C-4(2g) and C-4(2h)). Other characterization data were described in ref 16. 1-O-(2-Methoxy-4-carbonylphenyl)-β-D-glucuronic acid (2i): white solid, 25 mg, 100% yield; 1H NMR (400 MHz, CD3OD) δ 7.64 (s, 1H, Harom), 7.57 (d, J = 8.39 Hz, 1H, Harom), 7.18 (d, 1H, Harom), 5.04 (d, J1,2 = 7.35 Hz, 1H, H-1), 3.93 (s, 3H, OCH3), 3.82 (d, J4,5 = 9.29 Hz, 1H, H-5), 3.67−3.59 (m, 3H, H-2, H-3, and H-4); 13C NMR (100 MHz, CD3OD) δ 175.0 (CO), 173.3 (CO), 148.3 (Cqarom), 148.0 (Cqarom), 131.6 (Cqarom), 122.8 (Carom), 114.8 (Carom), 113.2 (Carom), 100.6 (C-1), 75.8 (C-3), 75.7 (C-5), 73.0 (C-2), 72.0(C-4), 55.5 (OCH3). Other characterization data were described in ref 16. 1-O-(2-Methoxy-5-carbonylphenyl)-β-D-glucuronic acid (2j): yellow solid, 78 mg, 100% yield; 1H NMR (400 MHz, D2O) δ 7.59−7.57 (m, 2H, Harom), 7.05 (d, J = 9.26 Hz, 1H, Harom), 5.10 (d, J1,2 = 8.17 Hz, 1H, H-1), 3.85−3.81 (m, 4H, CH2(Bn) and H-5), 3.58−3.50 (m, 3H, H-2, H-3, and H-4); 13C NMR (100 MHz, D2O) δ 175.4 (C O), 144.7 (Cqarom), 125.2 (Carom), 116.9 (Carom), 111.9 (Carom), 100.5 (C-1), 76.2 (C-3), 75.2 (C-5), 72.7 (C-4), 71.7 (C-2), 55.9 (OCH3). Other characterization data were described in ref 16.
separated, but knowing its exact proportion in the mixture, by proton NMR, allows them to be used as standards. The reaction conditions were optimized to minimize the formation of the disulfate byproduct, when more than one free hydroxyl group was present. All of the compounds were prepared in a 500 mg scale; however, this reproducible and reliable method can be easily scaled up. The methodology used for the synthesis of the glucuronyl donor was the same as that of Nakajima et al.17 The trichloroacetimidate 4 was the glycosyl donor for all of the glycosylation reactions described in Figure 4. Due to the unreactivity of the phenols as glycosyl acceptors, the yields were moderate. After 4 h, no more conversion was observed, the reactions were quenched, and the unreacted starting materials were recovered and reused. Attempts to improve the yield of these reactions were made. Using TMSOTf as the promotor instead of BF3·OEt2 did not afford better yields. The corresponding p-tolyl thioglycoside was also used as the glycosyl donor, with both TfOH/NIS and TMSOTf/NIS reaction systems; however, no improvement was observed. As expected, due to the participating effect of acetyl participating group at the 2-position of the sugar, only the β anomers were obtained. For the glycosylation reactions, contrary to the sulfate synthesis, the carboxylic acid functional group on the phenols had to be protected, in the form of a benzyl ester, which would be removed at the same time as the methyl ester at the C-6 position of the glucuronic acid. The purpose of using a benzyl ester as the protecting group of the phenolic carboxylic acids was to introduce a stereo effect induced by the bulkiness of the benzyl group when two free hydroxyl groups are present. In these cases, we opted for not protecting one of the hydroxyl groups, which avoided protection/deprotection reactions, shortening the synthesis, which compensates the lower yields for our purposes. As with the sulfate syntheses, when more than one product could be formed, except in the case of pyrogallol, the two possible isomers were obtained. Glycosylation with pyrogallol afforded exclusively the 2-O-glycoside 6b, and no 3-O-glycoside was isolated, even though the yield of 6b was only 17%. The glycosylation reaction with 4-methylcatechol 5c afforded the two isomers, 6d:6e 1:1.42; this proportion was confirmed and determined by NMR, analyzing the chemical shifts of the phenolic moiety, for this pair of compounds and for all other intermediates resulting from the deprotection reactions and the final compounds 2d and 2e. In the case of the obtainment of the protocatechuic acid glucuronides, the two isomers 6g:6h showed a proportion of 1:2.5, and their confirmation was equally based on the proton NMR spectra for all of the intermediates and final compounds. Removal of the acetates was accomplished by treating glycosylation products 6 with sodium methoxide in methanol, in very good to excellent yields (Figure 4). Finally, hydrolysis of the methyl and benzyl esters, when applicable, with lithium hydroxide in water afforded the final glucuronides. The results obtained for compounds 2f, 2g, 2h, 2i, and 2j were consistent with the characterization data from the literature.16 The data for compound 2a also were consistent with the literature;15 however, the synthesis strategy followed was different, and we were able to obtain the compound with better yields. In summary, using short and efficient syntheses, several phenolic metabolites were synthesized. These compounds were
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RESULTS AND DISCUSSION Seven sulfate derivatives (Figure 2) and 10 glucuronic acid derivatives (Figure 3) were synthesized, with the main aim of providing new standards for the analysis and study of phenol metabolization in complex biological samples such as plasma and urine. Sulfate derivatives were synthesized as we previously described.9 Reaction of phenols with sulfur trioxide−pyridine, in pyridine at 65 °C for 24 h, afforded the corresponding sulfates (Figure 3). The products were converted into the sodium salts, and their final yields are reported in Figure 4. This synthesis strategy was efficient and straightforward, allowing the obtainment of several different sulfates using simple manipulations. Any unreacted starting material was easily removed by washing the reaction mixture with ethyl acetate. The major challenge of the methodology was the elimination of traces of pyridine from the products, due to its high boiling point. In the case of phenols with more than one free, nonsymmetrical hydroxyl groups (3c, protocatechuic acid; and 3e, caffeic acid), a mixture of the two possible isomers was obtained. Protocatechuic acid afforded sulfates 1c and 1d in a proportion of 1:2.03. This proportion was determined by proton NMR, where the protons vicinal to the sulfate suffered a more drastic downfield chemical shift change. The same was observed for the products of the sulfation of caffeic acid, which afforded products 1f and 1g, in a 1:1.27 proportion. Interestingly, the same hydroxyl group (at the R2 position) was preferentially sulfated in both substrates; however, the proportion was not the same, the selectivity being higher in protocatechuic acid 3c. In both cases the two isomers were not F
DOI: 10.1021/acs.jafc.6b05629 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
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fully characterized. Their main application could be as standards for biological and analytical studies, where complex mixtures are studied. These derivatives will be crucial tools for the rigorous identification of the metabolites present in the biological samples. Furthermore, as the isolation of these metabolites is difficult and very time-consuming and they are obtained in small quantities, their chemical synthesis provides the compounds in larger quantities and with high purity for more specific tests, for the evaluation of their bioactivity. New synthetic compounds are particularly important to perform structure−activity relationship studies aimed at understanding the modes of action of the most biologically active compounds. Organic synthesis will also be important for the further modification of these phenol-derived metabolites to improve or modulate their biological activities. The described synthesis methods are simple and highly flexible to be used in the synthesis of sulfated and glucuronated derivatives of many other phenolic compounds. This is particularly important, as there is a great lack of standards of this family of compounds.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05629. 1 H NMR and 13C NMR of all new compounds (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(M.R.V.) Phone: +351 214 469 775. E-mail: rventura@itqb. unl.pt. ORCID
A. Filipa Almeida: 0000-0002-8399-0710 M. Rita Ventura: 0000-0002-6854-7278 Funding
We acknowledge FCT for financial support of CNS (IF/ 01097/2013). C.N.S. and A.F.A. also acknowledge funding via BacHBerry (Project FP7-613793; www.bachberry.eu). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge the Research Unit MOSTMICRO (UID/ CQB/04612/2013). The NMR spectrometers are part of The National NMR Facility, supported by FCT (RECI/BBBBQB/ 0230/2012). The iNOVA4Health Research Unit (LISBOA-010145-FEDER-007344), which is cofunded by Fundaçaõ para a Ciência e Tecnologia/Ministério da Ciência e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged.
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ABBREVIATIONS USED AcOEt, ethyl acetate; equiv, equivalent; BF3·OEt2, boron trifluoride diethyl etherate; NaHCO3, sodium bicarbonate; Na2SO4, sodium sulfate anhydrous; TMSOTf, trimethylsilyl trifluoromethanesulfonate; TfOH, trifluoromethanesulfonic acid; NIS, N-iodosuccinimide
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REFERENCES
(1) Tsao, R. Chemistry and biochemistry of dietary polyphenols. Nutrients 2010, 2, 1231−1246. G
DOI: 10.1021/acs.jafc.6b05629 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
