Synthesis of New Sulfated and Glucuronated Metabolites of Dietary Phenolic Compounds Identified in Human Biological Samples

Article abstract of DOI:10.1021/acs.jafc.6b05629

(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 metaboli

Full text of DOI:10.1021/acs.jafc.6b05629

Article
pubs.acs.org/JAFC
Synthesis of New Sulfated and Glucuronated Metabolites of Dietary
Phenolic Compounds Identified in Human Biological Samples
A. Filipa Almeida,†,‡ Claudia N. Santos,†,‡ and M. Rita Ventura*,‡
́
†Instituto de Biologia Experimental e Tecnolog
́
ica, Universidade Nova de Lisboa Instituto de Tecnologia Quimica e Biologica,
Apartado 12, 2781-901 Oeiras, Portugal
‡
́
Instituto de Tecnologia Quımica e Biolog
́
ica Anton
́
io Xavier, NOVAUniversidade Nova de Lisboa, Av. da Republica, 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
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 well-
characterized structure, it is very difficult to identify these
compounds from biological samples.
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, O-
glucuronates, 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
The bacterial arylsulfotransferase (AST) from Desulfitobacte-
rium 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 bio-
logical 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 glucuronida-
tion 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
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
Received: December 16, 2016
Revised: February 13, 2017
Accepted: February 15, 2017
Published: February 15, 2017
puree 9,11
́
.
However, for some new metabolites, with predicted
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.6b05629
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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.
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
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 (Sigma-
Aldrich) loaded with Na+. The column was eluted with water. The
final residue was dried under vacuum and characterized by 1H and 13
C
NMR.
1
Guaiacol-O-sulfate (1a): 644 mg, 78% yield; H 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).
1
Benzoic acid 4-O-sulfate (1b): 473 mg, 68% yield; H 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 (CO), 155.1
(Cqarom), 131.6 (Carom), 127.4 (Cqarom), 121.1 (Carom).
Protocatechuic acid 4-O-sulfate (1c) and protocatechuic acid 3-
O-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 (CO), 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)).
Figure 1. Structures of sulfate derivatives.
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
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
1
Gel Merck 60. H NMR spectra were obtained at 400 MHz in D2O,
Figure 2. Structures of glucuronate derivatives.
B
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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).
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-glucuro-
20
nate (6c): white solid, 248 mg, 54% yield; [α]D −46.0 (c 1.0,
Caffeic acid 4-O-sulfate (1f) and caffeic acid 3-O-sulfate (1g):
1
CH2Cl2); H NMR (400 MHz, CDCl3) δ 7.15 (dd, J = 7.19 Hz, J =
498 mg, 69% yield, mixture of both compounds in approximately equal
1
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
(CO), 169.4 (CO), 169.3 (CO), 167.0 (COsugar), 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, C-
4), 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-D-
glucuronate (6d) and methyl 2,3,4-tri-O-acetyl-1-O-(2-hydroxy-4-
methylphenyl)-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, H-
2(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) (CO), 169.7(4) (CO), 169.6(9) (CO), 169.4
(CO), 166.7(2) (COsugar), 166.6(9) (COsugar), 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 (C-
5(4e)), 72.5 (C-5(4d)), 71.5, 71.4, 71.1, 69.0(4), 69.0(2) (C-2(4e), C-
3(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-glu-
amounts (1:1.27) with a contamination of disulfate; H 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 (CO(3)), 171.0 (CO(4)), 150.5 (Cqarom), 148.2
(Cqarom), 145.5 (CHCH), 145.1 (CHCH), 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 stepsfirst, 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-glucuro-
nate (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 dichloro-
methane 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
1
(6a): yellow solid, 94 mg, 48% yield; H NMR (400 MHz, CDCl3) δ
20
curonate (6f): white solid, 204 mg, 36% yield; [α]D −21.67 (c 0.6,
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 (CO), 169.8
(CO), 169.4 (CO), 166.7 (COsugar), 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.
1
CH2Cl2); H 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 (CO), 169.2 (CO), 166.7 (CO), 165.7 (CO), 160.1
(CO), 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 (C-
1), 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-(1,3-dihydroxyphenyl)-β-D-glucuro-
20
nate (6b): white solid, 80 mg, 17% yield; [α]D −33.14 (c 0.7,
1
CH2Cl2); H NMR (400 MHz, CDCl3) δ 6.95 (t, J = 8.34 Hz, 1H,
Methyl 2,3,4-tri-O-acetyl-1-O-(2-hydroxy-4-benzylcarbonylphen-
yl)-β-D-glucuronate (6g) and methyl 2,3,4-tri-O-acetyl-1-O-(2-
hydroxy-5-benzylcarbonylphenyl)-β-D-glucuronate (6h): mixture of
both compounds in a proportion of 1:1.27; white solid, 114 mg, 24%
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 (CO), 169.4 (CO), 169.2 (CO), 166.3 (COsugar),
149.3 (Cqarom), 132.2 (Cqarom), 126.8 (Carom), 108.6 (Carom), 103.0 (C-
1
yield; H 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
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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
(CO), 169.4 (CO), 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 (C-
2, 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).
3.55 (m, 3H, H-2, H-3, and H-4); 13C NMR (100 MHz, D2O) δ 170.5
(CO), 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, H-
5), 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 (CO), 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):
Methyl 2,3,4-tri-O-acetyl-1-O-(2-methoxy-4-benzylcarbonyl-
phenyl)-β-D-glucuronate (6i): white solid, 220 mg, 37% yield;
20
[α]D −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
(CO), 169.3 (CO), 166.8 (CO), 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).
20
1
white solid, 100 mg, 100% yield; [α]D −77.50 (c 0.2, MeOH); H
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 (CO),
166.0 (CO), 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 (C-
5), 73.0 (C-2), 71.4 (C-4), 66.2 (CH2), 51.5 (OCH3).
Methyl 2,3,4-tri-O-acetyl-1-O-(2-methoxy-5-benzylcarbonyl-
phenyl)-β-D-glucuronate (6j): white solid, 217 mg, 48% yield;
20
1
[α]D +1.0 (c 0.3, CH2Cl2); H 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 (CO),
169.4 (CO), 169.2 (CO), 166.8 (CO), 165.6 (CO), 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).
Methyl 1-O-(2-hydroxy-4-benzoxycarbonylphenyl)-β-D-glucuro-
nate (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, H-
5(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 (CO), 166.1 (CO), 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 C-
4(7h)), 66.2 (CH2(7g)), 66.1 (CH2(7h)), 51.5 (OCH3).
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
Methyl 1-O-(2-methoxy-4-benzoxycarbonylphenyl)-β-D-glucuro-
nate (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)),
(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, H-
4), 3.58−3.48 (m, 2H, H-2 and H-3); 13C NMR (100 MHz, CD3OD)
δ 169.5 (CO), 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 (CO), 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).
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
(CO), 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-glucuro-
nate (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 H-
3); 13C NMR (100 MHz, CD3OD) δ 169.4 (CO), 166.0 (CO),
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).
Methyl 1-O-(2-methoxyphenyl)-β-D-glucuronate (7c): white solid,
20
1
33 mg, 46% yield; [α]D −81.17 (c 0.6, CH3OH); H 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
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Journal of Agricultural and Food Chemistry
Article
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.
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
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 (CO), 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 1-
O-(2-hydroxy-4-methylphenyl)-β-D-glucuronic acid (2e): white solid,
1
1
mg, 100% yield; H NMR (400 MHz, CD3OD) δ 7.26 (dd, J = 1.42
62 mg, 73% yield; H NMR (400 MHz, CD3OD) δ 7.13 (d, J = 8.05
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 (CO),
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,
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), H-
3
(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 (CO), 175.0
(CO), 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 (C-
1
(2e)), 103.7 (C-1(2d)), 76.1 (C-3), 75.3 (C-5), 73.3 (C-2), 72.2 (C-4),
20
1
54 mg, 91% yield; [α]D −63.27 (c 1.0, CH3OH); H 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 (CO), 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
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 (CO), 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.
mg, 80% yield; [α]D20 −85.99 (c 0.8, CH3OH); H NMR (400 MHz,
1
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
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Journal of Agricultural and Food Chemistry
Article
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 H-
5(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 (CO), 172.3 (CO),
171.8 (CO), 149.9 (Cqarom(2h)), 147.9 (Cqarom(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 character-
ization 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
1-O-(2-Methoxy-4-carbonylphenyl)-β-D-glucuronic acid (2i):
1
white solid, 25 mg, 100% yield; H 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); 13
C
NMR (100 MHz, CD3OD) δ 175.0 (CO), 173.3 (CO), 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): yel-
low 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.
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 manipu-
lations. 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

Journal of Agricultural and Food Chemistry
Article
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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
* Supporting Information
■
(9) Pimpao, R. C.; Ventura, M. R.; Ferreira, R. B.; Williamson, G.;
̃
S
Santos, C. N. Phenolic sulfates as new and highly abundant
metabolites in human plasma after ingestion of a mixed berry fruit
puree. Br. J. Nutr. 2015, 113, 454−63.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.6b05629.
1H NMR and 13C NMR of all new compounds (PDF)
(10) Ferrars, R. M.; Cassidy, A.; Curtis, P.; Kay, C. D. Phenolic
metabolites of anthocyanins following a dietary intervention study in
post-menopausal women. Mol. Nutr. Food Res. 2014, 58, 490−502.
AUTHOR INFORMATION
Corresponding Author
*(M.R.V.) Phone: +351 214 469 775. E-mail: rventura@itqb.
unl.pt.
(11) Pimpao, R. C.; Dew, T.; Figueira, M. E.; McDougall, G. J.;
̃
■
Stewart, D.; Ferreira, R. B.; Santos, C. N.; Williamson, G. Urinary
metabolite profiling identifies novel colonic metabolites and
conjugates of phenolics in healthy volunteers. Mol. Nutr. Food Res.
2014, 58, 1414−1425.
ORCID
(12) van der Horst, M. A.; van Lieshout, J. F. T.; Bury, A.; Hartog, A.
F.; Wever, R. Sulfation of various alcoholic groups by an arylsulfate
sulfotransferase from Desulfitobacterium hafniense and synthesis of
estradiol sulfate. Adv. Synth. Catal. 2012, 354, 3501−3508.
(13) van der Horst, M. A.; Hartog, A. F.; El Morabet, R.; Marais, A.;
Kircz, M.; Wever, R. Enzymatic sulfation of phenolic hydroxy groups
of various plant metabolites by an arylsulfotransferase. Eur. J. Org.
Chem. 2015, 2015, 534−541.
(14) Armarego, W. L. F.; Chai, C. Purification of Laboratory
Chemicals, 5th ed.; Elsevier, 2003.
(15) Turner, H. J.; Burton, M. Chromogenic substrates for β-D-
glucuronidase activity and use thereof for microbial detection. WO
2015/067926 A1, 2015.
(16) Zhang, Q.; Raheem, K. S.; Botting, N. P.; Slawin, A. M. Z.; Kay,
C. D.; O’Hagan, D. Flavonoid metabolism: the synthesis of phenolic
glucuronides and sulfates as candidate metabolites for bioactivity
studies of dietary flavonoids. Tetrahedron 2012, 68, 4194−4201.
(17) Nakajima, R.; Ono, M.; Aiso, S.; Akita, H. Synthesis of methyl 1-
O-(4-hydroxymethamphetaminyl)-α-D-glucopyranouronate. Chem.
Pharm. Bull. 2005, 53, 684−687.
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.
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-01-
0145-FEDER-007344), which is cofunded by Fundaca̧ o para a
̃
̂ ́ ̂
Ciencia e Tecnologia/Ministerio da Ciencia e do Ensino
Superior, through national funds, and by FEDER under the
PT2020 Partnership Agreement, is acknowledged.
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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DOI: 10.1021/acs.jafc.6b05629
J. Agric. Food Chem. XXXX, XXX, XXX−XXX