A concise synthesis of glucuronide metabolites of urolithin-B, resveratrol, and hydroxytyrosol

Article abstract of DOI:10.1016/j.carres.2009.05.016

A simple and direct strategy to chemically synthesize O-β-d-glucuronides of urolithin-B 4, resveratrol 5, and the corresponding hydroxytyrosol derivatives 6, 7 (as a regioisomeric mixture), and 8 is described. The critical glycosylation step has been opti

Full text of DOI:10.1016/j.carres.2009.05.016

Carbohydrate Research 344 (2009) 1340–1346
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
A concise synthesis of glucuronide metabolites of urolithin-B, resveratrol,
and hydroxytyrosol
*
Ricardo Lucas, David Alcantara, Juan Carlos Morales
Department of Bioorganic Chemistry, Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, 49 Américo Vespucio, 41092 Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 April 2009
Received in revised form 11 May 2009
Accepted 18 May 2009
Available online 22 May 2009
A simple and direct strategy to chemically synthesize O-b-D-glucuronides of urolithin-B 4, resveratrol 5,
and the corresponding hydroxytyrosol derivatives 6, 7 (as a regioisomeric mixture), and 8 is described.
The critical glycosylation step has been optimized using a structurally simple phenol, urolithin-B, by
modification of several reaction parameters (solvent, promoter, and glucuronide donor). Very high yields
have been obtained in the first synthesis of the O-b-D-glucuronide of urolithin-B 4. Extension of these
reaction conditions was used for the synthesis of resveratrol-3-O-glucuronide 5 where a higher yield than
previously reported was obtained by using the much more common trichloroacetimidate glucuronide
Keywords:
Resveratrol
Hydroxytyrosol
Urolithin-B
Glucuronic metabolites
Glycosylation reactions
Trichloroacetimidate donor
donor. Finally, three O-b-
D-glucuronides of hydroxytyrosol 6, 7, and 8 have been synthesized for the first
time using chemical synthesis.
Ó 2009 Elsevier Ltd. All rights reserved.
Polyphenols, plant secondary metabolites found in fruits, vege-
tables, and natural beverages, have been related to the lower inci-
dence of certain types of cancer, cardiovascular diseases or
inflammatory-related illnesses. Three of the most important die-
tary polyphenols are resveratrol, hydroxytyrosol, and ellagitannins.
trans-Resveratrol ((E)-3,40,5-trihydroxystilbene, 2, Fig. 1) is found
in plants, roots, peanuts, seeds, berries, and grapes. It has been
shown that resveratrol exhibits an impressive array of antioxida-
tive,1 anticarcinogenic,2 anti-inflammatory3, and cardioprotective
properties.4 Hydroxytyrosol (3,4-dihydroxyphenylethanol, 3), a
orthodiphenolic antioxidant found in olives and olive oil, has been
reported to show antibacterial,5 anti-inflammatory6, and antican-
cer activity,7 inhibition of human LDL oxidation,8 and prevention
of platelet aggregation.9 Ellagitannins, found in raspberries, pome-
granates, and other fruits, have shown to inhibit angiogenesis10
and possess anti-atherogenic properties.11 They are metabolized
in humans to ellagic acid, which is further metabolized by the hu-
man colonic microflora to dibenzopyran-6-one derivatives; mainly
urolithins A and B (see Fig. 1 for urolithin-B, 1).
cule.12,13 The major metabolic pathway found in vivo for these die-
tary phenols is O-conjugation via glucuronidation and sulfation.
Preparation of 3-O-b-D D-glucuronide conjugates of
- and 40-O-b-
trans-resveratrol has been reported by two different approaches.
Learmonth14 described the chemical synthesis of these two O-glu-
curonides, using a long route based on a Heck coupling of iodo-O-
b-D-glucuronate derivatives with the corresponding styrenes.
Wang et al.15 prepared the corresponding glucuronides by direct
coupling of resveratrol with the bromo glucuronide donor in a very
low yield, probably due to the low solubility of resveratrol in or-
ganic solvents. The same authors also used a second route by silyl
protection of the phenolic hydroxyl groups to finally couple a quite
uncommon trifluoroacetamidite glucuronide donor in moderate
yields. Biocatalyzed synthesis of hydroxytyrosol, tyrosol, homova-
nillic alcohol, and 3-(40-hydroxyphenyl)propanol glucuronides has
been developed.16 However, enzymatic synthesis requires the use
of either expensive glucuronyl transferases or liver microsomes
followed by a final tedious purification of the mixtures obtained.
Finally, to the best of our knowledge, the synthesis of urolithin glu-
curonic acid metabolites has not been described so far. In this work
we report a simple and direct strategy to chemically synthesize
Knowledge of the metabolic fate of these biologically relevant
dietary phenols is of fundamental importance to know their real
potential as health protecting agents or their possible use as drugs.
This aspect is especially significant since different studies have
pointed out that the biological activity of the metabolites of certain
polyphenols is similar to or higher than that of the parent mole-
O-b-D-glucuronides of urolithin-B 4, resveratrol 5, and the corre-
sponding hydroxytyrosol derivatives 6, 7 (as a regioisomeric mix-
ture), and 8.
First, we optimized the glycosylation reaction for a structurally
simple, but biologically relevant phenol, such as urolithin-B (1).
The preparation of the corresponding glucuronic derivative donors
9–11 (Scheme 1) is carried out from the commercially available
* Corresponding author. Tel.: +34 954 489561; fax: +34 954 460565.
E-mail address: jcmorales@iiq.csic.es (J.C. Morales).
D
-glucurono-6,3-lactone.17,18 The
a/b diastereomeric mixtures of
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.05.016

R. Lucas et al. / Carbohydrate Research 344 (2009) 1340–1346
1341
OH
O
O
HO
HO
HO
OH
HO
OH
1
2
3
O
OH
HO2C
O
O
HO2C
HO
O
O
HO
HO
HO
O
OH
OH
4
5
OH
HO2C
HO
HO
HO2C
HO
O
O
HO2C
HO
HO
OH
OH
O
OH
O
O
HO
OH
O
OH
HO
OH
HO
OH
6
7
8
Figure 1. Chemical structure of dietary phenolics and their glucuronide metabolites.
O
MeO2C
RO
O
O
NH
CCl3
+
RO
HO
O
OR
R=Ac
R=Bz
R=Piv 11
1
9
10
Glycosylation reaction
(see table 1)
Na2CO3, MeOH, H2O
O
O
MeO2C
RO
O
R= Ac
R= Bz
R= Piv
12
13
14
4
(87%)
R= H
O
RO
OR
Scheme 1. Synthesis of urolithin-B-O-b-
D
-glucuronides, protected 12–14.
trichloroacetimidate donors 9–11 were used without further puri-
fication. Urolithin-B was prepared by the condensation of resor-
cinol and 2-bromobenzoic acid with copper sulfate as catalyst in
alkaline medium (Hurtley reaction).19 Glycosylation of urolithin-
B acceptor 1 and glucuronosyl donor 11 was performed using
TMSOTf as the promoter (Scheme 1 and Table 1, entry 1). The ste-
reoselectivity of the reaction was very good and the b-diastereoiso-
mer 14 could be isolated in moderate yields. Attempts to improve
the yield of the reaction by modifying the solvent failed (Table 1,
entries 2 and 3). Then, we considered the use of BF3ꢀOEt2, the most
common promoter used in aromatic glycosylation.20 Pleasingly,
reaction of 1 with the glucuronosyl donor 11 produced compound
14 in much higher yield (78%, Table 1, entry 4). When the glucur-
onosyl donors 9 and 10 were reacted with urolithin-B (Table 1, en-
tries 5 and 6), products 12 and 13 were obtained in very high yields
(95% and 83%, respectively) with no sign of orthoester formation.
Deprotection of the acyl groups in compounds 12–14 was car-
ried out using Na2CO3 in a mixture MeOH–H2O to avoid the for-
Table 1
Glycosylation reactions with glucuronic donors
Entry
Acceptor
Donor
Promoter
Solvent
Product
Yielda (%)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
11
11
11
11
9
10
11
9
9
9
10
11
TMSOTf d
TMSOTf d
TMSOTf d
CH2Cl2
14
—
45
—
b
THFb or Et2Ob
CH3CNc
mation of the
a,b-unsaturated acid side product and ring
14
14
12
13
17
16
19,20
22
23
24
24
78
95
84
54
71
51
18
84
53
opening of the urolithin lactone that is observed when using
KOH or NaOH in aqueous alcohols.21 Reaction of the acetylated
derivative 12 gave urolithin-B glucuronide 4 in high yield whereas
a very slow reaction was observed for the benzoyl or pivaloyl
derivatives 13 and 14.
Next, we moved to the structurally more complex phenolic
derivative resveratrol 2. We decided to explore different glycosyl-
ation conditions (Scheme 2) with the alcohol acceptor 15 (22%
yield from resveratrol) used previously by Wang et al.15 Our initial
reaction conditions using glucuronosyl donor 11 in dry dichloro-
methane and using TMSOTf as promoter produced the protected
compound 17 in a 54% yield (Table 1, entry 7). When using our
optimized conditions, glucuronosyl donor 9 in dry dichlorometh-
ane and BF3ꢀOEt2 as the promoter, the corresponding protected res-
e
b
BF3ꢀOEt2
CH2Cl2
e
b
BF3ꢀOEt2
CH2Cl2
e
b
1
BF3ꢀOEt2
CH2Cl2
15
15
18
21
21
21
TMSOTf d
CH2Cl2
b
e
b
BF3ꢀOEt2
CH2Cl2
e
b
BF3ꢀOEt2
CH2Cl2
e
b
10
11
12
BF3ꢀOEt2
CH2Cl2
e
b
BF3ꢀOEt2
CH2Cl2
e
b
BF3ꢀOEt2
CH2Cl2
a
Yields of isolated products after purification.
Glycosylation reactions were carried out with 1.5 equiv of donor and 1.0 equiv
b
of acceptor, at ꢁ10 °C.
c
Glycosylation reaction was carried out at 0 °C to room temperature for 16 h.
0.05 equiv of promoter was used.
0.25 equiv of promoter was added.
d
e

1342
R. Lucas et al. / Carbohydrate Research 344 (2009) 1340–1346
OTBS
MeO2C
RO
RO
HO
O
NH
+
15
O
CCl3
OR
OTBS
9
R= Ac
R= Piv 11
Glycosylation reaction
(see table 1)
R2O2C
OR3
R1O
O
R1O
O
OR1
1. HF.Pyridine, THF
2. Na2CO3, MeOH, H2O
OR3
R1= Ac, R2=Me, R3=TBS
R1= Piv, R2=Me, R3=TBS
5 (67%, 2 steps)
R1,R2,R3= H
16
17
Scheme 2. Synthesis of protected resveratrol-3-O-b-D-glucuronide (16, 17) and resveratrol-3-O-b-D-glucuronide (5).
veratrol derivative 16 was obtained in a better yield than reported
previously (71%; Table 1, entry 8). Resveratrol derivative 16 was
deprotected in two steps (Scheme 2) by treatment with HFꢀPy in
THF and basic hydrolysis resulting in compound 5 (67%).
and BF3ꢀOEt2 as the promoter (Scheme 4). The low yield of the reac-
tion (18%; Table 1, entry 10) might be explained due to the low
reactivity of the glucuronosyl donor and the very reactive glycosyl
acceptor 21, which is very rapidly acetylated at the primary posi-
tion. No improvement was observed when TMSOTf was used as
the promoter or by changing the order of addition of the reagents.
Better yields of the glycosylated products (23 and 24) could be ob-
tained when the glycosylation reaction was carried out using the
benzoyl and the pivaloyl-protected glucuronosyl donors 10 and
11 (84% and 53%; Table 1, entries 11 and 12, respectively).
Deprotection of derivatives 22–24 was carried out in two steps.
First, Na2CO3 was used to remove the acetyl groups and then, tri-
fluoroacetic acid in a H2O–THF mixture to remove the acetal group.
The reaction of compounds 22 and 23 under these conditions pro-
duced the hydroxytyrosol glucuronide 8 in good yields (87% and
75%, respectively). In the case of the pivaloyl derivative 24, no
deprotection reaction was observed by treatment with Na2CO3 in
Hydroxytyrosol 3 was prepared by reduction of 3,4-dihydroxy-
phenyl acetic acid with lithium aluminum hydride in 75% yield.22
Then, enzymatic selective acetylation using the immobilized lipase
Novozym 435Ò produced the glycosyl acceptor 18 (95% yield).23,24
The glycosylation reaction of acceptor 18 with glucuronosyl donor
9 was performed using the same conditions described above
(Scheme 3) to give compounds 19 and 20 in a regioisomeric mix-
ture (51%; Table 1, entry 9). Deprotection of all the acetyl groups
under basic conditions (Scheme 3) afforded a 1.7:1 regioisomeric
mixture of 4- and 3-O-b-glucuronides of hydroxytyrosol 7 and 8.
The ratio between the regioisomers was calculated by 1H NMR. A
tri-O-acetyl glucuronide derivative of hydroxytyrosol (22) was pre-
pared by glycosylation of the ketal-protected hydroxytyrosol
2123,24 with the glucuronosyl donor 9 in dry dichloromethane
aqueous methanol, and partial formation of the
a,b-unsaturated
MeO2C
O
HO
HO
OAc
NH
CCl3
AcO
+
AcO
O
OAc
9
18
Glycosylation reaction
(see table 1)
MeO2C
AcO
HO
O
O
MeO2C
+
O
OAc
AcO
AcO
OAc
O
OAc
AcO
HO
19
OAc
20
Na2CO3, MeOH, H2O (88%)
HO2C
HO
O
O
+
HO2C
HO
HO
OH
O
HO
OH
O
OH
HO
HO
OH
6
7
Scheme 3. Synthesis of hydroxytyrosol 40-O-b- -glucuronide (6) and hydroxytyrosol 30-O-b-
D D-glucuronide (7).

R. Lucas et al. / Carbohydrate Research 344 (2009) 1340–1346
1343
1.2. Methyl-[1-(dibenzo[b,d]pyranyl-6-one)]-2,3,4-tri-O-acetyl-
MeO2C
AcO
AcO
HO
O
O
O
NH
CCl3
b-D
-glucopyranosyluronate (12)
+
O
To a solution of trichloroacetimidate 917 (450 mg, 0.943 mmol)
OAc
21
and urolithin-B 1,19 (100 mg, 0.472 mmol) in anhydrous CH2Cl2
(6 mL) at ꢁ10 °C, BF3ꢀOEt2 (0.235 mmol) was added dropwise. After
5 h, TLC (hexane–EtOAc 2:1) showed the formation of a major
product (Rf 0.23) and complete consumption of the starting mate-
rial. The reaction was quenched by the addition of NEt3 and con-
centrated in vacuo. The resulting residue was purified by flash
column chromatography (hexane–EtOAc from 3:1 to 1:1) to afford
9
R= Ac
R= Bz
10
Glycosylation reaction
(see table 1)
R= Piv 11
R2O2C
R1O
R1O
O
O
O
OR1
O
12 (238 mg, 95%) as a yellow glassy solid; ½a D22
ꢁ29.5 (c 1 in
ꢂ
R1= Ac, R2=Me
R1= Bz, R2=Me
R1= Piv, R2= Me
22
23
24
CHCl3); 1H NMR (300 MHz, CDCl3) d 8.30 (d, J = 8.1 Hz, Harom),
7.98–7.88 (m, 2H, Harom), 7.76, 7.75 (2t, J = 8.1 Hz, 2H, Harom), 6.9–
6.93 (m, 2H, Harom), 5.40–5.26 (m, 4H, H-1, H-2, H-3, H-4), 4.29
(d, J = 8.7 Hz, H-5), 3.73 (s, 3H, OCH3), 2.10, 2.05 (2s, 9H, CH3C@O);
13C NMR (62.5 MHz, CDCl3) d 170.0, 169.3, 169.2 (C@O), 161.01
(C@O lactone), 157.9, 152.1, 135.0, 134.4, 130.5, 128.3, 123.6,
121.3, 120.2, 114.1, 113.4, 105.0 (Carom), 98.4 (C-1), 72.5, 71.7,
70.8, 69.0 (C-2, C-3, C-4, C-5), 53.0 (CH3O), 20.6, 20.5 (CH3–C@O).
ESIMS: Calcd for C26H24NaO12: 551.1165. Found: 551.1165.
1. Na2CO3, MeOH, H2O
2. TFA, THF-H2O
HO2C
OH
OH
O
HO
HO
O
OH
8 (75%, 2 steps from 23)
Scheme 4. Synthesis of 2-(30,40-dihydroxyphenyl)ethanol-1-O-b-
1.3. Methyl-[1-(dibenzo[b,d]pyranyl-6-one)]-2,3,4-tri-O-
benzoyl-b-D-glucopyranosyluronate (13)
D-glucuronide (8).
To a solution of trichloroacetimidate 1017 (100 mg, 0.150 mmol)
and 1 (22 mg, 0.10 mmol) in anhydrous CH2Cl2 (3 mL) at ꢁ10 °C,
BF3ꢀOEt2 (0.0375 mmol) was added dropwise. After 5 h, TLC (hex-
ane–EtOAc 3:1) showed the formation of a major product (Rf
0.20) and complete consumption of the starting material. The reac-
tion was quenched by the addition of NEt3 and concentrated in va-
cuo. The resulting residue was purified by flash column
chromatography (toluene–EtOAc, from 10:1 to 6:1) to afford 13
acid side product was detected under reaction with NaOH in aque-
ous methanol.
In conclusion, a short and efficient chemical synthesis has
been developed for the stereoselective preparation of important
protected glucuronide metabolites 12–14, 16, 17, 19, 20, 22, 23,
and 24. The O-b-
been synthesized for the first time. The major glucuronic acid
metabolite of resveratrol, resveratrol 3-O-b- -glucuronide, 5, has
D-glucuronide conjugate of urolithin-B (4) has
D
(60 mg, 84%) as a yellow glassy solid; ½a D22
ꢂ
+31.0 (c 1 in CHCl3);
been prepared using the much more common trichloroacetimi-
date glycosyl donor obtaining a better yield of the product than
previously reported. Finally, three hydroxytyrosol metabolites 6,
7 (as a regioisomeric mixture), and 8 have been synthesized fol-
lowing similar glycosylation reaction conditions. It is important
to note that these phenolic glucuronides will help to elucidate
their actual contribution to the reported biological functions
in vivo in comparison with their parent compounds. These com-
pounds will also find use in the more accurate determination of
the metabolic and pharmacokinetic profiles of urolithins, resvera-
trol, and hydroxytyrosol.
1H NMR (300 MHz, CDCl3) d 8.36 (d, J = 8.1 Hz, Harom), 8.01–7.01
(m, 21H, Harom), 6.04 (t, J = 8.7 Hz, 1H, H-3), 5.92–5.84 (m, 2H, H-
2, H-4), 5.62 (d, J = 6.7 Hz, H-1), 4.62 (d, J = 8.7 Hz, H-5), 3.68 (s,
3H, OCH3); 13C NMR (62.5 MHz, CDCl3) d 170.0, 165.5, 165.2,
165.0 (C@O), 161.1 (C@O lactone), 152.1, 134.9, 134.6, 133.5,
133.4, 130.6, 129.9, 128.9, 128.8, 128.7, 128.6, 128.5, 128.4,
128.3, 123.9, 121.3, 120.3, 114.4, 113.5, 105.2 (Carom), 98.7 (C-1),
73.0 (C-5), 71.1 (C-3), 70.9 (C-2), 69.4 (C-4), 53.0 (CH3O). ESIMS:
Calcd for C41H30O12: 714.2. Found: 714.2.
1.4. Methyl-[1-(dibenzo[b,d]pyranyl-6-one)]-2,3,4-tri-O-
pivaloyl-b-
D
-glucopyranosyluronate (14)
1. Experimental
To
a
solution of trichloroacetimidate 1121,25 (260 mg,
1.1. General methods
0.43 mmol) and 1 (46.0 mg, 0.21 mmol) in anhydrous CH2Cl2
(6 mL) at ꢁ10 °C, BF3ꢀOEt2 (0.1075 mmol) was added dropwise.
After 2 h we observed a complete consumption of the starting
material. The reaction was quenched by the addition of NEt3 and
concentrated in vacuo. The resulting residue was purified by flash
column chromatography (hexane–EtOAc, from 8:1 to 2:1) to afford
14 (108 mg, 78%) as a yellow glassy solid; 1H NMR (300 MHz,
CDCl3) d 8.35 (d, J = 8.1 Hz, Harom), 8.00–7.96 (2d, J = 8.1 Hz, 2H,
Harom), 7.80, 7.54 (2t, J = 8.1 Hz, 2H, Harom), 6.98 (s, 2H, Harom),
5.50 (t, J = 9.1 Hz, 1H, H-4), 5.42–5.34 (m, 2H, H-2, H-3), 5.23
(d, J = 7.8 Hz, H-1), 4.29 (d, J = 9.1 Hz, H-5), 3.76 (s, 3H, OCH3),
1.16 (s, 27H, C(CH3)3); 13C NMR (62.5 MHz, CDCl3) d 177.0, 176.5,
176.4 (C@O), 166.6 (C@O lactone), 158.2, 152.1, 135.0, 134.5,
130.6, 128.4, 124.1, 121.3, 120.3, 114.1, 113.5, 105.0 (Carom), 99.1
(C-1), 72.9 (C-5), 71.2, 70.4, 69.0 (C-2, C-3, C-4), 53.0 (CH3O),
All chemicals were obtained from chemical suppliers and used
without further purification, unless otherwise noted. All reactions
were monitored by TLC on precoated Silica-Gel 60 plates F254,
and detected by heating with Mostain (500 mL of 10% H2SO4,
25 g of (NH4)6Mo7O24ꢀ4H2O, 1 g Ce(SO4)2ꢀ4H2O). Products were
purified by flash chromatography with Silica Gel 60 (200–400
mesh). NMR spectra were recorded on 300, 400 or 500 MHz NMR
equipment, at room temperature for solutions in CDCl3, D2O or
CD3OD. Chemical shifts are referred to the solvent signal and are
expressed in ppm. 2D NMR experiments (COSY, TOCSY, ROESY,
and HMQC) were carried out when necessary to assign the corre-
sponding signals of the new compounds. Sephadex G-25 then
ion-exchanged with Dowex 50W was used in the purification of
several glucuronic metabolites. Finally, samples were lyophilized
to dryness three times from D2O to deuterate all exchangeable
protons.
38.7 (C(CH3)3), 27.0 (C(CH3)3). ESIMS: Calcd for C35H42NaO12
677.2574. Found: 677.2569.
:

1344
R. Lucas et al. / Carbohydrate Research 344 (2009) 1340–1346
1.5. Sodium 1-(dibenzo[b,d]pyranyl-6-one)]-b-
syl-uronic acid (4)
D
-glucopyrano-
(C-5), 71.4 (C-3), 70.5 (C-2), 69.1 (C-4), 52.9 (CH3O), 38.8
(C(CH3)3), 27.4, 27.3, 27.2 (C(CH3)3), 25.9, 25.7 (SiC(CH3)3), 18.3,
18.2 (SiC(CH3)3), ꢁ4.19, ꢁ4.39 (Si(CH3)2). ESIMS: Calcd for
C48H74O12Si2: 899.4797. Found: 899.4827.
A suspension of ester 12 (40 mg, 0.075 mmol) in methanol
(1 mL) was stirred at 20° C with a solution of Na2CO3 (22 mg,
0.204 mmol) in H2O (0.5 mL). After 5 h, water (1 mL) was added,
followed by addition of glacial acetic acid to adjust the pH to 6.2.
The solvents were then removed and residue was purified by RP-
C18 column eluting with buffer triethylammonium acetate
10 mM and CH3CN (1:1). Fractions containing the desired product
were freeze-dried three times affording compound 4 (29 mg, 83%).
1.8. (E)-1-(5-Hydroxy-3-O-b-glucuronopyranosidophenyl)-2-
(40-hydroxyphenyl)-ethene (trans-resveratrol-3-O-b-
glucuronide) (5)
To a solution of 16 (700 mg, 0.9 mmol) in dry THF (15 mL) was
added (HF)ꢀPy (2.5 mL) at room temperature. The reaction was stir-
red for 6 h and was then diluted with EtOAc (50 mL) and NH4Cl sat
(5 mL) was added. The organic phase was washed with NH4Cl sat
(2 ꢃ 25 mL). The solvents were then removed in vacuo and the
crude was purified by flash column chromatography (hexane–
EtOAc from 4:1 to 1:1) to afford the corresponding desylilated
compound 5 (390 mg, 80%). The desylilated product (100 mg,
0.183 mmol) and Na2CO3 (52 mg, 0.49 mmol) were dissolved in a
solution of methanol (4 mL) and H2O (2 mL). The reaction mixture
was stirred at room temperature for 16 h. After this period, water
(1 mL) was added, followed by addition of glacial acetic acid to ad-
just the pH to 6.2. The solvents were then removed and residue
was purified by sephadex G-25 eluting with H2O–MeOH (9:1).
Fractions containing the desired product were freeze-dried afford-
1H NMR (500 MHz, D2O)
d 7.9 (d, J = 7.85 Hz, Harom), 7.31
(t, J = 7.7 Hz, 1H, Harom), 7.11 (t, J = 7.7 Hz, 1H, Harom), 6.97 (d, J =
8.8 Hz, 1H, Harom), 6.49 (d, 1H, J = 8.8 Hz, Harom), 6.22 (s, 1H,
Harom), 4.91 (d, J = 7.7 Hz, 1H, H-1), 3.86 (d, J = 9.3 Hz, 1H, H-4),
3.67–3.56 (m, 3H, H-2, H-3, H-5); 13C NMR (100.5 MHz, D2O) d
181.5 (C@O), 162.7 (C@O lactone), 157.6, 149.9, 135.5, 133.3,
128.9, 128.2, 123.6, 120.8, 117.2, 113.7, 111.7 (Carom), 99.6 (C-1),
76.7 (C-5), 75.3, 72.7, 71.9 (C-2, C-3, C-4). ESIMS: Calcd for
C19H15Na2O9: 433.0511. Found: 433.0493.
1.6. (E)-1-[3-tert-Butyldimethylsilyloxy-5-O-(2,3,4-tri-O-acetyl-
b-D
-glucopyranoside)phenyl]-2-(40-tert-butyldimethylsilyloxy-
phenyl) ethene methyl ester (16)
ing compound 5 (49 mg, 83%); ½a D22
ꢂ
ꢁ17.5 (c 1 in CHCl3); 1H NMR
To a solution of trichloroacetimidate 917 (1.77 g, 3.72 mmol)
and resveratrol derivative 1515 (1.7 g, 3.72 mmol) in anhydrous
(400 MHz, D2O) d 7.19 (d, J = 8.0 Hz, 2H, Harom), 6.80 (d, J = 16.4 Hz,
CH@CH), 6.66 (d, J = 8.0 Hz, 2H, Harom), 6.58 (d, J = 16.4 Hz, 1H,
CH@CH), 6.49 (d, J = 10.0 Hz, 2H, Harom), 6.30 (s, 1H, Harom), 4.89
(d, J = 7.2 Hz, 1H, H-1), 3.77 (d, J = 8.9 Hz, 1H, H-5), 3.56–3.52 (m,
3H, H-3, H-4, H-2); 13C NMR (100.5 MHz, D2O) d 175.5 (C@O),
163.1, 162.7, 161.3 158.3, 140.1, 129.0 (Carom), 128.2 (@CH),
126.3, 124.3, 117.5 (@CH), 109.6, 104.4, 103.3, 100.1 (C-1), 76.1
(C-5), 75.3 (C-3), 72.8 (C-2), 71.7 (C-4). ESIMS: Calcd for C20H19O8
(Mꢁ17): 387.10. Found: 387.00.
CH2Cl2 (30 mL) at ꢁ10 °C, BF3ꢀOEt2 (0.93 mmol, 930
lL) was added.
After 90 min, TLC showed the formation of a major product and
consumption of the glycosyl donor. The reaction was then
quenched by the addition of NEt3 and concentrated in vacuo. The
resulting residue was purified by flash column chromatography
(hexane–EtOAc, from 4:1 to 1:1) to afford 16 (2.0 g, 71%) as a yel-
low glassy solid; ½a D22
ꢂ
ꢁ19.5 (c 1 in CHCl3); 1H NMR (300 MHz,
CDCl3) d 7.15 (d, J = 8.0 Hz, Harom), 6.75 (d, J = 16.0 Hz, Harom),
6.64–6.46 (m, 6H, Harom, @CH), 6.17 (m, 1H, @CH), 5.15–5.06
(m, 3H, H-3, H-4, H-2), 4.93 (d, J = 7.2 Hz, 1H, H-1), 3.99 (d, J =
8.1 Hz, 1H, H-5), 3.52 (s, 3H, OCH3), 1.86, 1.82 (2s, 9H, CH3C@O),
0.76 (3s, 18H, C(CH3)3), ꢁ0.10 (s, 12H, Si–C(CH3)2); 13C NMR
(75 MHz, CDCl3) d 170.2, 169.4, 169.3, 166.9 (C@O), 157.7, 156.8,
155.7 (Cqarom), 139.9, 130.2, 129.2, 127.8, 127.7, 126.0, 120.3, 113.4
(Carom), 108.1 (@CH), 99.1 (C-1), 72.6 (C-5), 71.9 (C-3), 71.0 (C-2),
69.1 (C-4), 53.0 (CH3O), 25.7 (C(CH3)3), 20.6, 20.5 (C(CH3)3), 18.2
(SiC(CH3)3), ꢁ4.38 (Si/CH3)2). ESIMS: Calcd for C39H56NaO12Si2:
795.3234. Found: 795.3208.
1.9. 2-[30-Hydroxy-40-(methyl 2,3,4-tri-O-acetyl-b-
D-
glucopyranosyluronate)phenyl] EtOAc (19) and 2-[40-hydroxy-
30-(methyl 2,3,4-tri-O-acetyl-b-
D-glucopyranosyluronate)
phenyl] EtOAc (20)
To a solution of trichloroacetimidate 917 (366 mg, 0.76 mmol)
and 2-(3,4-dihydroxyphenyl)-EtOAc 1823,24 (200 mg, 1.02 mmol)
in anhydrous CH2Cl2 (6 mL) at ꢁ10 °C, BF3ꢀOEt2 (25
lL, 0.19 mmol)
was added dropwise. After 2 h, TLC (hexane–EtOAc 2:1) showed
the formation of a new product and complete consumption of
the glycosyl donor. The reaction was neutralized with NEt3 and
concentrated in vacuo. The resulting residue was purified by flash
column chromatography (hexane–EtOAc from 3:1 to 1:1) to afford
a regioisomeric mixture of 19 and 20 (205 mg, 51%); 1H NMR
(400 MHz, CDCl3) d 6.89–6.80 (m, 3H, Harom), 6.63 (m, 1H, Harom),
6.19 (m, 2H, Harom), 5.37–5.25 (m, 6H, H-2a, H-2b, H-3a. H-3b, H-
4a, H-4b), 5.03, 5.02 (2d, J = 7.6 Hz, 2H, H-1a, H-1b), 4.23–4.17
(m, 6H, H-5a, H-5b, 2 ꢃ CH2), 3.74 (s, 6H, CH3O), 2.84–2.80 (m,
4H, 2 ꢃ CH2), 2.09–2.06 (m, 24H, CH3C@O); 13C NMR (100.5 MHz,
CDCl3) d 171.1, 171.0 (COOCH3), 170.0, 169.8, 169.7 169.4, 166.8,
143.9, 142.8, 135.4, 130.0, 125.7, 120.6, 118.3, 118.0, 117.0,
116.5, 101.4, 100.1 (C-1a, C-1b), 72.4, 71.5, 71.4, 71.2, 71.1, 69.0,
68.9, 64.8, 64.7, 53.1, 34.5, 34.2, 20.9, 20.6, 20.5, 20.4. ESIMS: Calcd
for C23H28NaO13Na: 536.1. Found: 536.8
1.7. (E)-1-[3-tert-Butyldimethylsilyloxy-5-O-(2,3,4-tri-O-
pivaloyl-b-D
-glucopyranoside)phenyl]-2-(40-tert-
butyldimethylsilyloxy-phenyl) ethene methyl ester (17)
To a solution of trichloroacetimidate 11 (100 mg, 0.165 mmol)
and resveratrol derivative 15 (120 mg, 0.262 mmol) in anhydrous
CH2Cl2 (2 mL) at ꢁ10 °C, TMSOTf (0.016 mmol, 3.0
lL) was added.
After a 3-h period, TLC showed the formation of a major product
and consumption of the glycosyl donor. The reaction was then
quenched by the addition of NEt3 and concentrated in vacuo. The
resulting residue was purified by flash column chromatography
(hexane–EtOAc from 10:1 to 8:1) to afford 17 (80 mg, 54%) as a yel-
low glassy solid; 1H NMR (300 MHz, CDCl3) d 7.38 (d, J = 8.4 Hz, 2H,
Harom), 6.98 (d, J = 16.0 Hz, 1H, Harom), 6.86–6.39 (m, 6H, Harom
,
@CH), 5.48–5.33 (m, 3H, H-3, H-4, H-2), 5.14 (d, J = 7.8 Hz, 1H, H-
1), 4.24 (d, J = 9.9 Hz, 1H, H-5), 3.76 (s, 3H, OCH3), 1.23, 1.18,
1.17 (3s, 27H, C(CH3)3), 1.01, 1.00 (2s, 18H, SiC(CH3)3), 0.23, 0.20
(2s, 12H, Si–C(CH3)2); 13C NMR (75 MHz, CDCl3) d 177.0, 176.5,
176.4, 166.8 (C@O), 157.9, 156.8, 155.7, 139.9, 130.3, 129.2,
128.0, 127.8, 126.1, 120.3, 113.4, 108.1, 107.9, 99.7 (C-1), 73.0
1.10. 2-[30-Hydroxy-40-b-
D-glucopyranosyluronic acid)phenyl]
ethanol (6) and 2-[40-hydroxy-30-b-
D-glucopyranosyluronic acid)
phenyl] ethanol (7)
A solution of the regioisomeric mixture of 19 and 20 (60 mg,
0.11 mmol) in methanol (2 mL) was stirred at room temperature

R. Lucas et al. / Carbohydrate Research 344 (2009) 1340–1346
1345
with a solution of Na2CO3 (22 mg, 0.204 mmol) in H2O (0.5 mL).
After 16 h., water (1 mL) was added, followed by addition of glacial
acetic acid to adjust the pH to 6.2. The solvents were then removed
and residue was purified by sephadex G-25 eluting with H2O–
MeOH (9:1). Fractions containing the desired product mixture
were freeze-dried affording compounds 6 and 7 (32 mg, 88%) as
a 1.7:1 regioisomeric mixture; 1H NMR (500 MHz, D2O) d 7.00–
6.70 (m, 6H, Harom), 4.97, 4.94 (2d, J = 7.0 Hz, 2H, H-1, H-10),
3.77–3.75 (m, 2H, H-3, H-30), 3.76–3.52 (m, 10H, H-4, H-40, H-5,
H-50, CH2, H-2, H-20), 2.68–2.64 (m, 4H, CH2); 13C NMR (75 MHz,
D2O) d 181.1 175.0 (C@O), 143.8, 143.0, 135.1, 131.9, 124.3,
121.3, 117.4, 117.0, 116.9, 116.4 (Carom), 101.3, 101.0 (C-1, C-10),
76.3, 75.2, 72.6, 71.7, 62.5, 62.4, 46.5, 37.0. ESIMS: Calcd for
C14H15O9 (M3ꢁ): 327.1. Found: 327.0.
1H, H-3), 5.26 (t, J = 9.6 Hz, 1H, H-4), 5.09 (dd, J = 8.0 and 9.6 Hz,
1H, H-2), 4.59 (d, J = 8.0 Hz, 1H, H-1), 4.07 (d, J = 9.6 Hz, 1H, H-5),
4.03 (m, 1H, OCH2), 3.75 (s, 3H, CH3O), 3.67–3.61 (m, 1H, OCH2),
2.80–2.77 (m, 2H, PhCH2), 1.67, 1.66 (2s, 6H, C(CH3)2), 1.21, 1.17,
1.13 (3s, 27H, C(CH3)3); d (100 MHz, CDCl3); 170.0 (COOCH3),
169.2, 169.7 167.2 (C@O), 147.0, 145.0 (Cqarom), 130.9, 121.2,
117.6, 109.1, 107.9, 101.0 (C-1), 72.9, 71.6, 71.1, 69.4, 52.7, 38.7,
35.8, 27.1, 27.0, 26.9, 25.8. ESIMS: Calcd for C33H48O12Na:
659.3102. Found: 659.3043.
1.14. 2-Ethyl-O-(30,40dihydroxyphenyl)methyl-b-
D-
glucopyranosyluronate (8)
A solution of compound 23 (120 mg, 0.17 mmol) in methanol
(6 mL) was stirred at room temperature with a solution of Na2CO3
(110 mg, 1.02 mmol) in H2O (2.0 mL). After 4 days, water (1 mL)
was added, followed by addition of glacial acetic acid to adjust
the pH to 6.2. The solvents were then removed and residue was
used for the next step without further purification. The latter crude
was dissolved in THF–H2O (1:1, 2 mL) and TFA (3 mL) was then
added. The reaction mixture was stirred at room temperature for
48 h. Solvents were then removed in vacuo and the residue was
purified by Sephadex G-25 eluting with H2O–MeOH (9:1) and
RP-C18 eluting with H2O–CH3CN (from 100:0 to 70:30). Fractions
containing the desired product were freeze-dried affording com-
pound 8 (42 mg, 75%). 1H NMR (300 MHz, D2O) d 6.59–6.44 (m,
3H, Harom), 4.19 (d, J = 7.8 Hz, 1H, H-1), 3.79–3.74 (m, 1H, CH2),
3.62–3.52 (m, 2H, CH2, H-5), 3.31–3.20 (m, 2H, H-3, H-4), 3.05–
2.99 (m, 1H, H-2), 2.56–2.51 (m, 2H, CH2); d (75 MHz, D2O);
163.2, 162.7, 143.8, 142.2, 131.3, 121.1, 116.6, 116.1, 102.1 (C-1),
75.2, 72.7, 71.3, 71.1, 34.3. ESIMS: Calcd for C14H15O9 (M3ꢁ):
327.0733. Found: 327.0408.
1.11. 2-Ethyl-O-(2,2-dimethylbenzo[1,3]dioxol-5-yl)methyl-
2,3,4-tri-O-acetyl-b-D-glucopyranosyluronate (22)
To a solution of trichloroacetimidate 917 (986 mg, 0.76 mmol)
and the hydroxytyrosol derivative 2126 (200 mg, 1.02 mmol) in
anhydrous CH2Cl2 (8 mL) at ꢁ10 °C, BF3ꢀOEt2 (66
lL, 0.51 mmol)
was added dropwise. After a 2-h period the reaction was neutralized
with NEt3 and concentratedin vacuo. The resultingresidue was puri-
fied by flash column chromatography (toluene–EtOAc, from 6:1 to
3:1) to afford 22 (100 mg, 18%); 1H NMR (300 MHz, CDCl3) d 6.41–
6.33 (m, 3H, Harom), 5.01–4.98 (m, 2H, H-3, H-4), 4.82–4.79 (m, 1H,
H-2), 4.30 (d, J = 7.7 Hz, 1H, H-1), 3.89–3.77 (m, 2H, H-5, CH2), 3.53
(s, 3H, CH3O), 3.39 (m, 1H, CH2), 2.58–2.52 (m, 2H, CH2), 1.83, 1.79
(2s, 9H, CH3C@O), 1.42 (s, 6H, C(CH3)2); 13C NMR (75 MHz, CDCl3) d
170.1 (COOCH3), 169.3, 169.2, 169.7 167.2 (C@O), 147.3, 145.9
(Cqarom), 131.4, 121.2, 117.6, 109.2, 107.9, 100.7 (C-1), 72.6, 72.0,
71.1, 69.5, 52.9, 35.5, 29.7, 25.8, 20.6, 20.5, 20.4. ESIMS: Calcd for
C24H30O12Na: 533.2. Found: 533.2.
Acknowledgments
1.12. 10-(Methyl 2,3,4-tri-O-benzoyl-b-
D-glucopyranosyluronate)-
20[(30,40-isopropylidene)phenyl] ethanol (23)
We would like to thank JAE-Doc program (R.L.) and Intramural
Frontier Projects (200680F0132) from CSIC for financial support.
To a solution of trichloroacetimidate 10 (680 mg, 1.02 mmol)
and the hydroxytyrosol derivative 21 (90 mg, 0.46 mmol) in anhy-
drous CH2Cl2 (4 mL) at ꢁ10 °C, BF3ꢀOEt2 (66
lL, 0.51 mmol) was
Supplementary data
added dropwise. After 1 h, the reaction was neutralized with
NEt3 and concentrated in vacuo. The resulting residue was purified
by flash column chromatography (toluene–EtOAc from 20:1 to 6:1)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2009.05.016.
to afford 23 (270 mg, 84%); ½a D22
ꢂ
+25.2 (c 1 in CHCl3); 1H NMR
(300 MHz, CDCl3) d 7.89–7.28 (m, 15H, Harom), 6.57–6.40 (m, 3H,
Harom), 5.92 (t, J = 9.3 Hz, 1H, H-3), 5.72 (t, J = 9.6 Hz, 1H, H-4),
5.09 (dd, J = 7.5 and 9.3 Hz, 1H, H-2), 4.90 (d, J = 7.5 Hz, 1H, H-1),
4.36 (d, J = 9.6 Hz, 1H, H-5), 4.15 (m, 1H, OCH2), 3.74–3.71 (m,
4H, OCH2, CH3O), 2.82–2.77 (m, 2H, PhCH2), 1.64, 1.63 (2s, 6H,
C(CH3)2). 13C NMR (75 MHz, CDCl3) d 167.4, 165.6, 165.2, 165.0
(C@O), 147.3, 145.8 (Cqarom), 133.4, 133.3, 133.2, 131.2, 129.8,
129.0, 128.9, 128.8, 128.7, 128.4, 128.3, 128.2, 125.3, 121.2,
117.5, 109.1, 107.9, 101.0 (C-1), 72.9, 72.1, 71.1, 70.2, 52.9, 35.6,
25.8. ESIMS: Calcd for C39H36O12Na: 719.2104. Found: 719.2097.
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