Chemical synthesis of hydroxycinnamic acid glucosides and evaluation of their ability to stabilize natural colors via anthocyanin copigmentation

Article abstract of DOI:10.1021/jf071205v

This work describes the chemical synthesis of O-aryl-β-D-glucosides and 1-O-β-D-glucosyl esters of hydroxycinnamic acids. In particular, O-aryl-β-D-glucosides were efficiently prepared via a simple diastereoselective glycosylation procedure using phase transfer conditions. Despite the lability of its ester linkage, 1-O-β-D-caffeoylglucose could also be obtained using a Lewis acid catalyzed glycosylation step and a set of protective groups that can be removed under neutral conditions. Hydroxycinnamic acid O-aryl-β-D-glucosides were then quantitatively investigated for their affinity for the naturally occurring anthocyanin malvin (pigment). Formation of the π-stacking molecular complexes (copigmentation) was characterized in terms of binding constants and enthalpy and entropy changes. The glucosyl moiety did not significantly alter these thermodynamic parameters, in line with a binding process solely involving the polyphenolic nuclei.

Full text of DOI:10.1021/jf071205v

J. Agric. Food Chem. 2007, 55, 7573−7579 7573
Chemical Synthesis of Hydroxycinnamic Acid Glucosides and
Evaluation of Their Ability To Stabilize Natural Colors via
Anthocyanin Copigmentation
STEPHANIE GALLAND, NATHALIE MORA, MARYLINE ABERT-VIAN,
NJARA RAKOTOMANOMANA, AND OLIVIER DANGLES*
UMR408 Se´curite´ et Qualite´ des Produits d’Origine Ve´ge´tale, INRA, Universite´ d’Avignon,
33 rue Louis Pasteur, 84029 Avignon, France
This work describes the chemical synthesis of O-aryl-â-D-glucosides and 1-O-â-D-glucosyl esters of
hydroxycinnamic acids. In particular, O-aryl-â-D-glucosides were efficiently prepared via a simple
diastereoselective glycosylation procedure using phase transfer conditions. Despite the lability of its
ester linkage, 1-O-â-D-caffeoylglucose could also be obtained using a Lewis acid catalyzed
glycosylation step and a set of protective groups that can be removed under neutral conditions.
Hydroxycinnamic acid O-aryl-â-D-glucosides were then quantitatively investigated for their affinity for
the naturally occurring anthocyanin malvin (pigment). Formation of the π-stacking molecular complexes
(copigmentation) was characterized in terms of binding constants and enthalpy and entropy changes.
The glucosyl moiety did not significantly alter these thermodynamic parameters, in line with a binding
process solely involving the polyphenolic nuclei.
KEYWORDS: Hydroxycinnamic acid; glucoside; glucosyl ester; synthesis; copigmentation; anthocyanin
INTRODUCTION
routes to prepare hydroxycinnamic acid glucosides and glucosyl
esters. In addition, their ability to enhance natural colors by
interacting with anthocyanin plant pigments is investigated.
The benefits of a regular consumption of fruits and vegetables
in the prevention of cancers, cardiovascular diseases, age-related
neurodegeneration, and diabetes could be partially related to
the high content in polyphenols displayed by these foods (1-
4). Hydroxycinnamic acids are one of the most abundant classes
of dietary polyphenols and are generally found as esters of quinic
acid and tartaric acid and also as D-glucose conjugates (O-aryl-
â-D-glucosides, O-â-D-glucosyl esters) (5-10), especially in
berries and tomato.
Interestingly, the glucose moiety of some dietary flavonoids
has been shown to allow their uptake by intestinal cells (1-3)
according to two distinct mechanisms (11): deglucosylation by
the enzyme lactase phlorizin hydrolase and subsequent passive
diffusion of the aglycones through the enterocyte layer or active
transport via a D-glucose transporter present in the membrane
of intestinal cells and subsequent deglucosylation by a cytosolic
â-glucosidase. Similarly, the glucosylation of hydroxycinnamic
acids could increase their bioavailability.
MATERIALS AND METHODS
All starting materials were obtained from commercial suppliers and
were used without purification. Solvents were distilled over CaCl2,
CaH2, KOH, or NaOH. TLC analyses were performed on silica gel 60
F254 or C-18 silica gel F254s. Detection was achieved by UV light (254
nm) and by charring after exposure to a 5% H2SO4 solution in EtOH.
Purifications were performed by column chromatography on silica gel
60 (40-63 µm). Dowex 50Wx4-50 or Amberlite IRC 50 ion-exchange
resin was used for acidification. Melting points were measured on a
Barnstead Electrothermal 9100 apparatus and are uncorrected.
NMR. 1H and 13C NMR spectra were recorded on an Avance DPX-
300 Brucker apparatus at 300.13 MHz (1H) or 75.46 MHz (13C). NMR
chemical shifts are in parts per million relative to tetramethylsilane
using the deuterium signal of the solvent (CDCl3, CD3OD) for
1
calibration. H-1H coupling constants are in hertz.
HR-MS. High-resolution mass analyses were carried out on Qstar
Elite instrument (Applied Biosystems SCIEX, Foster City, CA)
equipped with API. Mass detection was performed in the negative or
positive electrospray ionization mode.
HPLC-MS. HPLC-MS analyses were performed on a HP1050
apparatus coupled to a UV-visible diode array detector and to a
Micromass Platform LCZ 4000 mass spectrometer. Mass detection was
performed in the negative electrospray ionization mode with a capillary
voltage of 25 V, a desolvation temperature of 80 °C, and a nitrogen
flow rate of 300 L/h. A 150 × 4.6 mm Altima C18 column (Alltech,
Deerfield, IL) equipped with a 7.5 × 4.6 mm precolumn was used for
chromatographic separations at 35 °C. The solvent system was a
Derivatives of hydroxycinnamic acids may be difficult to
extract in quantities required for biological or chemical studies,
especially identification and titration in plants, role in color
expression, and investigation of their facilitated intestinal
absorption. Hence, their chemical synthesis is an interesting
alternative. In this work, we report on novel and simple synthetic
* Author to whom correspondence should be addressed [telephone (33)
490 14 44 46; fax (33) 490 14 44 41; e-mail Olivier.Dangles@
univ-avignon.fr].
10.1021/jf071205v CCC: $37.00
© 2007 American Chemical Society
Published on Web 08/08/2007

7574 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Galland et al.
gradient of A (0.05% aqueous HCO2H) and B (MeCN) with 10% B at
0 min and 100% B at 20 min for O-aryl-â-D-glucosides and 0% B at
0 min and 90% B at 10 min for 1-O-â-D-caffeoylglucose (flow rate )
1 mL/min).
UV-Vis Spectroscopy. Copigmentation experiments were per-
formed on a Hewlett-Packard 8452A diode array UV-vis spectrometer
equipped with a magnetically stirred quartz cell (optical path length )
1 cm). The temperature in the cell was controlled by means of a water
thermostated bath.
Copigmentation. A 2 × 10-3 M solution of malvin was prepared
in MeOH acidified by concentrated HCl to a final concentration of 0.2
M. This solution was diluted in a pH 2.5 phosphate buffer (0.1 M H3-
PO4 + 1 M NaOH) to a final malvin concentration of 10-4 M. To a
portion of the latter solution was added 5 × 10-3 M copigment. The
malvin and malvin+copigment solutions were then mixed in various
proportions to prepare solutions of intermediate copigment concentra-
tions. The solutions were equilibrated for 20 min before spectroscopic
measurements. All calculations were performed at the isosbestic point
of malvin and its copigmentation complex, which was determined from
strongly acidic malvin and malvin+copigment solutions (copigment/
malvin molar ratio ) 50). The pH 2.5 malvin and malvin+copigment
solutions (copigment/malvin molar ratio ) 50) were then used for
investigating the temperature dependence of copigmentation in the range
of 25-40 °C.
J ) 9.9 Hz, 5.4 Hz, 2.4 Hz, H5′), 4.17-4.30 (2 dd, 2, J ) 12.3, 5.4
Hz, J ) 12.3, 2.4 Hz, 2 H6′), 5.13-5.33 (m, 4, H2′, H3′, H4′, H1′), 6.35
(d, 1, J ) 15.9 Hz, HR), 7.00 (d, 2, J ) 8.7 Hz, H3, H5), 7.47 (d, 2, J
) 8.7 Hz, H2, H6), 7.65 (d, 1, J ) 15.9 Hz, Hâ); 13C NMR (CDCl3), δ
21.01-21.07 (4, OCOCH3), 52.10 (CO2CH3), 62.29-70.22-71.47-
72.56-73.02 (5, C2′, C3′, C4′, C5′,C6′), 98.94 (C1′), 117.16 (CR), 117.48
(2, C3, C5), 130.00 (3, C1, C2, C6), 144.30 (Câ), 158.61 (C4), 167.89-
170.48 (4, OCOCH3), 170.54 (CO2CH3).
Methyl 4-(2′,3′,4′,6′-tetra-O-acetyl-â-D-glucopyranosyl) ferulate (2b):
crystallization in hexane/EtOAc gave 2b as a white amorphous powder;
yield, 65%; mp, 137 °C; Rf (hexane/EtOAc, 4:6), 0.56; 1H NMR
(CDCl3), δ 2.10 (m, 12, OCOCH3), 3.86 (s, 3, CO2CH3), 3.90 (s, 3,
OCH3), 3.91 (m, 1, H5′), 4.24-4.33 (2dd, 2, J ) 12.3, 5.1 Hz, J )
12.3, 1.8 Hz, H6′), 5.19-5.36 (m, 4, H1′, H2′, H3′, H4′), 6.40 (d, 1, J )
15.9 Hz, HR), 7.10 (m, 3, H2, H5, H6), 7.67 (d, 1, J ) 15.9 Hz, Hâ); 13
C
NMR (CDCl3), δ 21.41-21.48 (4, OCOCH3), 52.51 (CO2CH3), 56.85
(OCH3), 62.67-69.10-71.88-72.88-73.27 (5, C2′, C3′, C4′, C6′, C5′),
101.08 (C1′), 112.19 (C2), 117.92 (C5), 120.31 (CR), 122.42 (C6), 131.70
(C1), 144.99-148.59-151.52 (3, Câ, C3, C4), 168.15-171.05 (4,
OCOCH3), 171.35 (CO2CH3).
Methyl 4-(2′,3′,4′,6′-tetra-O-acetyl-â-d-glucopyranosyl) caffeate (2c):
column chromatography (SiO2; hexane/EtOAc, 6:4 to 1:1) gave 2c as
a white amorphous powder; yield, 30%; mp, 50 °C; Rf (hexane/EtOAc,
1
4:6), 0.52; H NMR (CDCl3), δ 2.07 (m, 12, OCOCH3), 3.81 (s, 3,
Data Analysis. The curve fittings were carried out on a PC using
the Scientist program (MicroMath, Salt Lake City, UT). Curve fittings
were achieved through least-squares regression. Standard deviations
are reported.
Synthesis. General Methylation Procedure. Hydroxycinnamic acid
(10.54 mmol) was dissolved in MeOH (30 mL) containing ca. 1 mL
of concentrated H2SO4. The solution was refluxed for about 24 h. After
concentration under reduced pressure, the solution was diluted with
EtOAc and washed with 5% aqueous NaHCO3 and water, then dried
over anhydrous Na2SO4, and concentrated. The raw product was purified
by crystallization in hexane/EtOAc.
Methyl p-coumarate (1a): yield, 90%; white amorphous powder; mp,
138-139 °C; Rf (hexane/EtOAc, 1:1), 0.65; 1H NMR (300 MHz,
CDCl3), δ 3.82 (s, 3, CO2CH3), 6.30 (d, 1, J ) 15.9 Hz, HR), 6.85 (d,
2, J ) 8.7 Hz, H3, H5), 7.45 (d, 2, J ) 8.7 Hz, H2, H6), 7.65 (d, 1, J
) 15.9 Hz, Hâ); 13C NMR (CDCl3), δ 52.21 (CO2CH3), 115.27 (CR),
116.34 (C3, C5), 128.50 (C1), 130.5 (C2, C6), 145.44 (Câ), 158.52 (C4),
168.79 (CO2CH3).
CO2CH3), 3.94 (m, 1, H5′) 4.15-4.28 (2 dd, 2, J ) 12.1, 5.0 Hz, J )
12.1, 1.4 Hz, H6′), 5.01 (d, 1, J ) 2.0 Hz, H1′), 5.19-5.36 (m, 3, H2′,
H3′, H4′), 6.35 (d, 1, J ) 15.9 Hz, HR), 6.91-7.16 (m, 3, H2, H5,H6),
7.59 (d, 1, J ) 15.9 Hz, Hâ); 13C NMR (CDCl3), δ 21.50-21.31 (4,
OCOCH3), 52.46 (CO2CH3), 62.41-68.86-72.12 72.85-73.13 (5, C2′,
C3′, C4′, C5′, C6′), 101.72 (C1′), 115.86-117.83-118.29-121.76 (4, CR,
C2, C5, C6), 132.28 (C1), 144.71-146.37-148.14 (3, C3, C4, Câ),
170.83-171.28 (4, OCOCH3), 171.33 (CO2CH3).
4-â-d-Glucopyranosylcoumaric Acid (3a). Compound 2a (150 mg,
0.46 mmol) was added to a mixture of 1 M NaOH (8 equiv)/H2O/
MeOH, 1:2:3 v/v/v (22 mL), and stirred for 8 h at room temperature.
Then, the mixture was acidified to pH 1-3 (wet pH paper) with Dowex
50 (H+ form) and concentrated under reduced pressure. Crystallization
in hexane/EtOAc afforded 3a as a white powder: yield, 90%; mp, 193
1
°C; Rf (C-18 silica, H2O + 0.05% HCO2H in MeCN, 1:1), 0.91; H
NMR (CD3OD), δ 3.38-3.54 (m, 4, H2′, H3′, H4′, H5′), 3.71-3.91 (2dd,
2, J ) 12.0, 1.8 Hz, J ) 12.0, 5.4 Hz, H6′), 4.99 (d, 1, J ) 7.5 Hz,
H1′), 6.37 (d, 1, J ) 15.9 Hz, HR), 7.14 (d, 2, J ) 8.7 Hz, H3, H5), 7.57
(d, 2, J ) 8.7 Hz, H2, H6), 7.65 (d, 1, J ) 15.9 Hz, Hâ); 13C NMR
(CD3OD), δ 61.96 (C6′), 70.31-73.84-76.43-76.95-77.23 (5, C1′,
C2′, C3′, C4′, C5′), 116.86 (CR), 116.97 (2, C3, C5), 129.703 (2, C2, C6),
130.54 (C1), 159.80 (Câ), 169.71 (C4), 178.52 (CO2H). HRMS-ESI,
m/z [M - H]- calcd for C15H17O8, 325.0929; found, 325.0934.
Methyl 4-â-d-Glucopyranosylferulate (3b′). Compound 2b (710 mg,
1.92 mmol) was dissolved in dry MeOH (80 mL) and treated with a
catalytic amount of MeONa. After 4 h of stirring, the mixture was
acidified with Amberlite IRC 50 (H+ form) to pH 1-3 (wet pH paper),
filtered, and evaporated. Crystallization in Et2O/MeOH yielded 3b′ as
a white solid: yield, 95%; mp, 173 °C; Rf (C-18 silica, 0.05% HCO2H
in MeCN, 1:1), 0.54; 1H NMR (CD3OD), δ 3.35-3.56 (m, 4, H2′, H3′,
H4′, H5′), 3.70-3.87 (2 dd, 2, J ) 12.3, 1.8 Hz, J ) 12.3, 5.1 Hz, H6′),
3.79 (s, 3, CO2CH3), 3.91 (s, 3, OCH3), 4.98 (d, 1, J ) 7.2 Hz, H1′),
6.47 (d, 1, J ) 15.9 Hz, HR), 7.19 (m, 2, H5, H6), 7.27 (d, 1, J ) 2.1
Hz, H2), 7.65 (d, 1, J ) 15.9 Hz, Hâ); 13C NMR (CD3OD), δ 51.46
(CO2CH3), 56.11 (OCH3), 61.82 (C6′), 70.63-74.16-77.22-77.66 (4,
C2′, C3′, C4′, C5′), 101.55 (C1′), 111.78 (C2), 116.42-116.73 (2, CR,
C5), 122.88 (C6), 130.08 (C1), 145.45-149.48-150.40 (3, C3, C4, Câ),
168.73 (CO2CH3).
Methyl ferulate (1b): yield, 70%; yellow amorphous powder; mp,
1
65 °C; Rf (hexane/EtOAc, 1:1), 0.39; H NMR (CDCl3), δ 3.82 (s, 3,
CO2CH3), 3.95 (s, 3, OCH3), 6.30 (d, 1, J ) 15.9 Hz, HR), 6.94 (d, 1,
J ) 8.1 Hz, H5), 7.04 (d, 1, J ) 1.8 Hz, H2), 7.09 (dd, J ) 8.1 Hz, 1.8
Hz, H6), 7.65 (d, 1, J ) 15.9 Hz, Hâ); 13C NMR (CDCl3), δ 52.02
(CO2CH3), 56.30 (OCH3), 109.91 (C2), 115.24 (C5), 115.42 (CR), 123.40
(C6), 127.27 (C1), 145.46 (Câ), 147.29 (C4), 148.50 (C3), 168.25 (CO2-
CH3).
Methyl caffeate (1c): yield, 77%; white powder; mp, 158 °C; Rf
(hexane/EtOAc, 1:1), 0.65; 1H NMR (CDCl3), δ 3.81 (s, 3, CO2CH3),
6.29 (d, 1, J ) 15.9 Hz, HR), 6.90 (d, 1, J ) 8.1 Hz, H5), 7.03 (dd, 1,
J ) 8.1 Hz, 1.8 Hz, H6), 7.10 (dd, 1, J ) 1.8 Hz, H2), 7.60 (d, 1, J )
15.9 Hz, Hâ); 13C NMR (CDCl3), δ 51.16 (CO2CH3), 114.78 (C2),
114.87 (C5), 115.95 (CR), 122.16 (C6), 127.15 (C1), 145.34 (Câ), 148.25
(C4), 167.58 (C3), 206.59 (CO2CH3).
General Glycosylation Procedure. A mixture of tetra-O-acetyl-R-
D-glucopyranosyl bromide (1.5 equiv) in anhydrous CH2Cl2 (25 mL)
was slowly added to a solution of methyl cinnamate (5.6 mmol) and
tris[2-(2-methoxyethoxy)ethyl]amine (1.5 equiv) in 20 mL of 1 M
NaHCO3/1 M KCl, 1:1. The mixture was refluxed for 48 h under N2.
After the addition of H2O (50 mL) and extraction with CH2Cl2 (3 ×
20 mL), the combined organic phases were successively washed with
1 M HCl (2 × 50 mL) and H2O (2 × 50 mL), dried over Na2SO4,
concentrated under reduced pressure, and purified by crystallization
or chromatography.
4-â-d-Glucopyranosylferulic Acid (3b). Compound 3b′ (100 mg, 0.27
mmol) was added to a mixture of 1 M NaOH (2 equiv)/H2O/MeOH,
1:2:3 v/v/v (3.2 mL), and stirred for 8 h at room temperature. Then,
the mixture was acidified to pH 1-3 (wet pH paper) with Dowex 50
(H+ form) and concentrated under reduced pressure. Crystallization in
hexane/EtOAc afforded 3b as a white powder: yield, 85%; mp, 199
°C; Rf (C-18 silica, 0.05% HCO2H in MeCN, 1:1), 0.88; 1H NMR (CD3-
OD), δ 3.36-3.56 (m, 4, H2′, H3′, H4′, H5′), 3.71-3.87 (2dd, 2, J )
12.0, 1.5 Hz, J ) 12.0, 5.1 Hz, H6′), 3.90 (s, 3, OCH3), 4.99 (d, 1, J )
Methyl 4-(2′,3′,4′,6′-tetra-O-acetyl)-â-D-glucopyranosyl coumarate
(2a): crystallization in hexane/EtOAc gave 2a as a white amorphous
1
powder; yield, 75%; mp, 162 °C; Rf (hexane/EtOAc, 4:6), 0.59; H
NMR (CDCl3), δ 2.00 (m, 12, OCOCH3), 3.80 (s, 3, OCH3), 3.90 (ddd,

Anthocyanin Copigmentation
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7575
7.2 Hz, H1′), 6.41 (d, 1, J ) 15.9 Hz, HR), 7.16 (dd, 1, J ) 9.0, 1.5 Hz,
H6), 7.20 (d, 1, J ) 9.0 Hz, H5), 7.27 (d, 1, J ) 1.5 Hz, H2), 7.63 (d,
1, J ) 15.9 Hz, Hâ); 13C NMR (CD3OD), δ 56.10 (OCH3), 61.82 (C6′),
70.63-74.17-77.22-77.65 (4, C2′, C3′, C4′, C5′), 101.58 (C1′), 111.78
(C2), 116.76-117.23 (2, C5, CR), 122.77 (C6), 129.99 (C1), 145.43 (Câ),
149.37-150.39 (2, C3, C4), 170.00 (CO2H). HRMS-ESI, m/z [M -
H]- calcd for C16H20O9, 355.1035; found, 355.1031.
Methyl 4-(â-d-Glucopyranosyl)caffeate (3c′). Deacetylation (same
procedure as for 2b′) yielded 3c′ as a white powder after crystallization
in Et2O/MeOH: yield, 80%; mp, 207 °C; Rf (C-18 silica, 0.05% HCO2H
in MeCN, 1:1), 0.67; 1H NMR (CD3OD), δ 3.47-3.53 (m, 4, H2′, H3′,
H4′, H5′), 3.73-3.92 (2dd, 2, J ) 12.1, 1.8 Hz, J ) 12.1, 4.8 Hz, H6′),
3.79 (s, 3, CO2CH3), 4.86 (d, 1, J ) 7.2 Hz, H1′), 6.40 (d, 1, J ) 15.9
Hz, HR), 7.06 (dd, 2, J ) 8.5, 2.1 Hz, H6), 7.12 (d, 1, J ) 2.1 Hz, H2),
7.21 (d, 1, J ) 8.5 Hz, H5), 7.59 (d, 1, J ) 15.9 Hz, Hâ); 13C NMR
(CD3OD), δ 52.87 (CO2CH3), 65.71 (C6′), 74.60-78.10-80.87-81.71
(4, C2′, C3′, C4′, C5′), 106.20 (C1′), 119.23 (C2), 120.35-121.48-125.47
(3, C5, C6, CR), 134.39 (C1), 149.40-151.90-152.19 (3, C3, C4, Câ),
172.65 (CO2CH3).
4-â-d-Glucopyranosylcaffeic Acid (3c). 3c was obtained by saponi-
fication of 3c′ in 1 M NaOH (3 equiv)/H2O/EtOH, 1:2:3 v/v/v: yield,
85%; mp, 135 °C; Rf (C-18 silica, 0.05% HCO2H in MeCN, 1:1), 0.92;
1H NMR (CD3OD), δ 3.47-3.59 (m, 4, H2′, H3′, H4′, H5′), 3.75-3.94
(dd, 2, J ) 12.0, 1.6 Hz, J ) 12.0, 4.6 Hz, H6′), 4.88 (d, 1, J ) 7.2 Hz,
H1′), 6.34 (d, 1, J ) 15.9 Hz, HR), 7.06 (dd, 1, J ) 8.4 Hz, J ) 1.9 Hz,
H6), 7.13 (d, 1, J ) 1.9 Hz, H2), 7.22 (d, 1, J ) 8.4 Hz, H5), 7.58 (d,
1, J ) 15.9 Hz, Hâ); NOE correlation between H5 and H1′; 13C NMR
(CD3OD), δ 61.76 (C6′), 70.65 (C4′), 74.16-76.91-77.76 (3, C2′, C3′,
C5′), 102.91 (C1′), 114.61-115.17-117.49 (3, CR, C2, C5), 121.41 (C6),
130.87 (C1), 134.25 (Câ), 147.84-148.01 (2, C3, C4), 168.59 (CO2H).
HRMS-ESI, m/z [M - H]- calcd for C15H18O9, 341.0879; found,
341.0873.
1,2,3,4,6-Penta-O-chloroacetyl-â-d-glucopyranoside (4). To a sus-
pension of D-glucose (2 g, 11.1 mmol) in CH2Cl2/pyridine, 9:1 (50
mL) was added dropwise chloroacetyl chloride (8.8 mL, 111.1 mmol)
at room temperature. The mixture was stirred to nearly complete
solubilization. Ice water (0 °C) was poured into the solution, and the
organic layer was separated and washed, respectively, with 1 M HCl,
saturated NaHCO3 and NaCl solution. The mixture was then dried over
Na2SO4 and concentrated under reduced pressure. The syrupy residue
was purified by column chromatography (SiO2, cyclohexane/EtOAc,
7:3 v/v) to give 4 in 65% yield: 1H NMR (CDCl3), δ 4.03-4.14 (5s,
10, COCH2Cl), 4.08-4.30 (m, 4, H2, H4,H5, H6), 5.22-5.49 (m, 1,
H3), 5.70 (d, 0.45, J ) 8.2 Hz, H1â), 6.31 (d, 0.55, J ) 1.9 Hz, H1R).
2,3,4,6-Tetra-O-chloroacetyl-â-D-glucopyranose (5). A mixture of
4 (2.7 g, 4.8 mmol) and hydrazinium acetate (0.44 g, 4.8 mmol) in
THF (50 mL) was stirred for 3 h at room temperature. After
concentration to dryness, the resulting oil was purified by column
chromatography (SiO2, cyclohexane/EtOAc, 7:3 v/v) to give 5 in 70%
yield: 1H NMR (CDCl3), δ 4.09-4.15 (m, 10, 2 × H6, 4 × COCH2-
Cl), 4.21-4.24 (m, 1, H5), 4.86-5.53 (m, 4, H1, H2, H3, H4).
2,3,4,6-Tetra-O-chloroacetyl-R-D-glucopyranosyltrichloroacetim-
idate (6). To a solution of 5 (2.7 g, 5.5 mmol) in CH2Cl2 (50 mL)
were added trichloroacetonitrile (5.57 mL, 55 mmol) and a catalytic
amount of 1,8-diazabicyclo[5.4.0]undec-7-ene. The mixture was stirred
for 4 h at room temperature. After concentration to dryness, the resulting
oil was purified with column chromatography (SiO2, cyclohexane/
EtOAc, 8:2 v/v) to give 6 in 60% yield: 1H NMR (CDCl3), δ 4.03-
4.14 (m, 8, 4 × COCH2Cl), 4.32-4.40 (m, 3, H5, 2 × H6), 5.25-5.35
(m, 2, H2, H4), 5.70 (t, 1, J ) 9.8 Hz, H3), 6.63 (d, 1, J ) 3.7 Hz, H1),
8.79 (s, 1, NH).
7.34-7.44 (m, 15, HAr), 7.63 (d, 1, J ) 15.9 Hz, Hâ); 13C NMR
(CDCl3), δ 70.62-71.33-71.71 (3, benzylic CH2), 114.12-114.71 (2,
C2, C5), 116.23 (CR), 123.44 (C6), 127.61-129.00 (16, C1, CBn-H),
136.65-137.23 (3, CBn-C), 145.42 (Câ),149.31-151.52 (2, C3, C4),
167.44 (CO).
3,4-Di-O-benzylcaffeic Acid (8). A mixture of 7 (2 g, 4.44 mmol)
in 3 M methanolic KOH (50 mL) was stirred for 12 h under reflux.
After cooling, the mixture was acidified with 3 M HCl. The precipitate
was collected and purified by crystallization in MeOH to afford 8 in
90% yield: mp, 196 °C; 1H NMR (CDCl3), δ 5.18-5.19 (2s, 4, benzylic
CH2), 6.32 (d, 1, J ) 15.9 Hz, HR), 7.06 (d, 1, J ) 8.4 Hz, H5), 7.16
(dd, 1, J ) 1.8, 8.4 Hz, H6), 7.30 (d, 1, J ) 1.8 Hz, H2), 7.31-7.50
(m, 10, HAr), 7.57 (d, 1, J ) 15.9 Hz, Hâ).
Compound 9. To a solution of 8 (0.33 g, 0.91 mmol) and 6 (0.87 g,
1.37 mmol) in anhydrous CH2Cl2 (under N2, molecular sieves 4A) was
added AgOTf (0.23 g, 0.91 mmol). The mixture was stirred for 1 h at
room temperature. After filtration on Celite, the solution was washed
with saturated NaHCO3 and water, dried over Na2SO4, and concentrated
under reduced pressure. The syrupy residue was purified with column
chromatography (SiO2, cyclohexane/EtOAc, 3:1 v/v) to give 9 in 50%
yield: 1H NMR (CDCl3), δ 3.91 (ddd, 1, J ) 2.1, 4.5, 9.9 Hz, H5′),
4.04-4.16 (4s, 8, COCH2Cl), 4.15 (dd, 1, J ) 12.3, 2.1 Hz, H6′), 4.34
(dd, 1, J ) 12.3, 4.5 Hz, H6′), 5.26-5.31 (m, 7, benzylic CH2, H2′, H3′,
H4′), 5.86 (d, 1, J ) 7.8 Hz, H1′), 6.23 (d, 1, J ) 15.9 Hz, HR), 6.94 (d,
1, J ) 8.4 Hz, H5), 7.10 (dd, 1, J ) 8.4, 1.8 Hz, H6), 7.15 (d, 1, J )
1.8 Hz, H2), 7.31-7.50 (m, 10, HAr), 7.66 (d, 1, J ) 15.9 Hz, Hâ).
Compound 10. Compound 9 was dissolved in a pyridine/water
mixture (1:1 v/v, pH 6.7) and stirred at room temperature for 10 h.
After concentration under reduced pressure, the syrupy residue was
purified with column chromatography (SiO2, EtOAc/MeOH, 95:5 v/v)
to give 10 in 80% yield: 1H NMR (CDCl3), δ 3.40-3.47 (m, 4, H2′,
H3′, H4′, H5′), 3.69-3.90 (m, 2, H6′), 5.18-5.19 (2s, 4, benzylic CH2),
5.61 (d, 1, J ) 7.1 Hz, H1′), 6.42 (d, 1, J ) 15.9 Hz, HR), 7.03-7.49
(m, 13, HAr, H2, H6, H5), 7.72 (d, 1, J ) 15.9 Hz, Hâ).
1-O-â-D-Caffeoylglucose (11). To a solution of 10 (50 mg, 0.095
mmol) in EtOH (10 mL) were added 1,4-cyclohexadiene (153 mg, 1.9
mmol) and 10% palladium on charcoal (20 mg) at room temperature.
The reaction was monitored by HPLC analysis. After the reaction was
complete, the mixture was filtered on Celite and the solvent was
evaporated under reduced pressure. The syrupy residue was purified
by flash chromatography (SiO2, 1% HCO2H in EtOAc) to give 11 in
40% yield: 1H NMR (CDCl3), δ 3.39-3.83 (m, 6, 2H6′, H2′, H3′, H4′,
H5′), 5.58 (d, 1, J ) 8.0 Hz, H1′), 6.34 (d, 1, J ) 16.0 Hz, HR), 6.87 (d,
1, J ) 8.0 Hz, H6), 7.07 (d, 1, J ) 8.0 Hz, H5), 7.13 (s, 1, H2), 7.68 (d,
1, J ) 16.0 Hz, Hâ); 13C NMR (CDCl3), δ 68.82-69.63-72.24-
75.49-77.01 (5, C2′, C3′, C4′, C5′, C6′), 94.14 (C1′), 113.45 (CR), 115.62
(C2), 115.75 (C4), 116.49 (C6), 122.79 (C3), 123.21 (C5), 126.30 (C1),
148.15 (Câ), 167.84 (CO). HRMS-ESI, m/z [M + H]+ calcd for
C15H18O9, 343.1023; found, 343.1024.
RESULTS AND DISCUSSION
Synthesis of Hydroxycinnamic Acid Glucosides (Figure
1). The key step is the glycosylation of methyl hydroxycin-
namates by 1-bromo-2,3,4,6-tetra-O-acetyl-R-glucopyranose
under phase-transfer conditions according to procedures previ-
ously used for the synthesis of flavonoid glucosides (12, 13).
The phase-transfer agent tris[2-(2-methoxyethoxy)ethyl]amine
(TMEA), which specifically binds K+, allows the transfer of
the phenolate ion from the alkaline aqueous solution to CH2Cl2
and its subsequent nucleophilic substitution with the glycosyl
bromide. The conditions had to be optimized to allow the
deprotonation of methyl hydroxycinnamates while minimizing
the hydrolysis of the glycosyl bromide or its conversion into
the corresponding glucal (14) or orthoester. Using 0.1 M
NaHCO3/0.1 M KCl or saturated K2CO3 aqueous solutions, we
obtained only moderate yields of glucosides (20-30%). The
choice of 1 M NaHCO3/1 M KCl led to substantial improve-
ments with yields of 30-75%. In the case of methyl caffeate,
3,4-Di-O-benzylcaffeic Acid Benzyl Ester (7). To a solution of caffeic
acid (1.5 g, 8.3 mmol) in acetone (30 mL) were added anhydrous K2-
CO3 (3.45 g, 25 mmol) and benzyl bromide (3.95 mL, 33.3 mmol).
The mixture was stirred for 12 h under reflux. Water (200 mL) was
poured into the solution, and the mixture was extracted with EtOAc,
dried over Na2SO4, and concentrated under reduced pressure. The
syrupy residue was purified by crystallization in EtOH to afford 7 in
1
85% yield: mp, 71 °C; H NMR (CDCl3), δ 5.18-5.21-5.25 (3s, 6,
benzylic CH2), 6.31 (d, 1, J ) 15.9 Hz, HR), 6.93 (d, 1, J ) 8.4 Hz,
H5), 7.08 (dd, 1, J ) 1.8, 8.4 Hz, H6), 7.14 (d, 1, J ) 1.8 Hz, H2),

7576 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Galland et al.
Figure 1. Synthesis route to hydroxycinnamic glucosides.
mixtures of 3′-O-â-D-glucoside, 4′-O-â-D-glucoside, and 3′,4′-
O-â-D-diglucoside were obtained. Only the major 4′-O-â-D-
glucoside was isolated by chromatography. As expected from
the R configuration of glycosyl bromide and the possible
orienting effect of the 2-O-acetyl group, the glycosidation was
stereoselective and led to the exclusive formation of the â
anomers [J(H1-H2) ) 7.5 Hz]. Less expectedly, the final
deprotection step (cleavage of the acetyl groups and methyl
ester) required rather extensive optimization by HPLC-MS
analysis in terms of the selected base and its concentration as
well as the temperature and duration of the reaction. For
instance, attempts at full deprotection using 1 M KOH in EtOH
were generally not satisfying and led to partially deprotected
glucosides and substantial degradation. Whereas 4-O-â-D-
glucopyranosylcoumaric acid (3a) could be directly obtained
from 2a using 1 M NaOH/H2O/EtOH (15), 2b and 2c had to
be deprotected in two steps involving sugar deacetylation by
mild catalytic methanolysis followed by methyl ester saponifica-
tion using 1 M NaOH/H2O/EtOH. 4-O-â-D-Glucopyranosylferu-
lic acid (3b) and 4-O-â-D-glucopyranosylcaffeic acid (3c) were
thus obtained in 80-85% overall yield.
Figure 2. Synthesis route to 1-O-â-D-caffeoylglucose.
known to increase the â stereoselectivity (20). In fact, both
procedures resulted in the migration (transesterification) of one
phenolic acyl group to the anomeric OH group of 5 with
retention of the R configuration. Finally, we selected the more
stable benzyl group for the protection of the phenolic OH groups
of caffeic acid because it has been reported that its cleavage by
mild hydrogenolysis using 1,4-cyclohexadiene as a hydrogen
donor can be achieved without hydrogenation of a carbon-
carbon double bond (21). 3,4-Di-O-benzylcaffeic acid (8) was
prepared by perbenzylation of caffeic acid followed by saponi-
fication of the benzyl ester in an overall yield of 90%. To ensure
a good â diastereoselectivity in the glucosylation step, hemiketal
5 was activated to the corresponding trichloroacetimidate (6)
and coupled to 8 in the presence of silver triflate in dry CH2Cl2
(22). Protected caffeoylglucose 9 was obtained in 60% yield as
a pure â anomer [J(H1-H2) ) 7.8 Hz] after purification by
chromatography. By contrast, activation of the trichloroace-
timidate by BF3/Et2O (23) or trimethylsilyltriflate and acylation
conditions using 3,4-di-O-benzylcaffeoyl chloride and 5 all led
to anomeric mixtures. Then, the four chloroacetyl groups of 9
were removed selectively in pyridine/H2O to yield 10 in 80%
yield. Finally, the palladium-catalyzed hydrogenolysis of the
two benzyl groups by 1,4-cyclohexadiene in EtOH at room
temperature was carefully monitored by HPLC-MS over 5 h,
that is, until complete consumption of 10. The major product,
1-O-â-D-caffeoylglucose (11), was then isolated by flash chro-
All glucosides were characterized by MS and NMR analyses.
The characteristics of 3a and 3b are in agreement with those
reported in the literature with compounds isolated from flaxseed
(7) or Riesling wine (16). Glucosylation at position 4′ of 3c
was confirmed by 1D-NOE experiments.
Synthesis of 1-O-â-D-Caffeoylglucose (Figure 2). The main
challenges were the development of a route allowing the
selective synthesis of the â-anomer and the choice of protecting
groups on the sugar and caffeic moieties that can be removed
under mild conditions, preserving the vinyl group and glucosyl
ester bond. The chloroacetyl group was chosen for the protection
of the sugar OH groups because it can be removed under neutral
conditions in which the glycosyl ester is expected to be stable
and also because it allows a 1,2-trans diastereoselectivity in the
glucosylation step (17). The preparation of the glucosyl donor
(5, pure R configuration) was achieved by chloroacetylation of
D-glucose with chloroacetyl chloride in CH2Cl2/pyridine fol-
lowed by anomeric deacylation by hydrazinium acetate in THF.
Different protecting groups were tested for the phenolic groups
of caffeic acid such as the chloroacetyl and acetyl groups (18).
Both were found to be too labile under the acylation conditions
used in the glucosylation step, that is, DCC/DMAP (19) or the
acyl chloride method in presence of triethylamine, which is
1
matography in 40% yield. Its structure was confirmed by H
and 13C NMR and high-resolution mass spectrometry.
Anthocyanin Copigmentation. Colorless polyphenols play
an important role in natural color expression by interacting with
anthocyanin pigments in the vacuoles of epidermal plant cells
(24-26). In this process called copigmentation, the colorless
polyphenol (copigment) stacks on the colored forms of the
anthocyanin, that is, the flavylium cation or the quinonoid bases
it forms upon deprotonation of its most acidic OH groups. The
visible absorption band of the copigmentation complex is usually
shifted to larger wavelengths with respect to the free colored
form (bathochromic effect). Moreover, in the mildly acidic
conditions of the vacuoles (typical pH in the range of 2.5-
7.5), the colored forms are thermodynamically unstable and are
converted to colorless forms via a process the key step of which

Anthocyanin Copigmentation
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7577
Figure 3. Hydration equilibrium of malvin (3,5-di-O-â-D-glucopyranosyl-
malvidin).
is the reversible water addition at C-2 of the flavylium cation
(AH+) and subsequent formation of a colorless hemiketal (B)
(thermodynamic constant Kh) (Figure 3) (27). Unlike B, AH+
and the quinonoid bases display a flat π-electron-rich aglycone
that is prone to developing π-stacking interactions with the
copigment molecule, so that copigmentation can be approxi-
matively rationalized as the selective interaction of the copig-
ment with the colored forms of the anthocyanin. Through the
copigmentation equilibrium (binding constant K), the copigment
molecule competes with water for the flavylium ion with the
overall effect that a fraction of hemiketal B is dehydrated to
AH+ due to its binding to the copigment (ligand L) to form
AHL+ (28-30). Thus, the color of the solution is enhanced
through a hyperchromic effect.
In this work, malvin (3,5-di-O-â-D-glucopyranosylmalvidin),
a common commercially available anthocyanin, was mixed with
various concentrations of the hydroxycinnamic acids and their
4-O-â-D-glucosides for a quantitative evaluation of the stability
of the corresponding copigmentation complexes. The choice of
the pH 2.5 phosphate buffer can be justified as follows: (1) at
this pH, free malvin is dominantly under the colorless B form
[a pH variation in the range of 0.6-3.0 allowed us to re-evaluate
the pKh value of malvin: pKh ) 1.52 ((0.02) at 25 °C (10
points, r ) 0.993)], thus providing a strong potential for color
regeneration; (2) at this pH, the sole colored form to consider
is the flavylium ion [first pKa ca. 4 (30)]. Color enhancement
via copigmentation of malvin by one of the chemically
synthesized copigments (4-â-D-glucopyranosylferulic acid) is
represented in Figure 4.
Absorbance values for calculations are collected at the
wavelength of the isosbestic point of AH+ and AHL+ deter-
mined in strongly acidic solutions (pH <1) at which malvin is
under a pure flavylium form. By definition, at the isosbestic
point, AH+ and AHL+ display the same molar absorption
coefficient, ꢀ. Combining the expressions of the thermodynamic
constants [Kh ) h[B]/[AH+] with h ) 10-pH, K ) [AHL+]/
([AH+][L])] with the laws of pigment and copigment conserva-
tion (C ) [AH+] + [B] + [AHL+], Lt ) [L] + [AHL+] ≈ [L]
because Lt . C) and Beer’s law [A ) ꢀ([AH+] + [AHL+]), A0
) ꢀ[AH+] in the absence of copigment, Ar0ef ) ꢀC ) absor-
bance of a strongly acidic solution of malvin under a pure
flavylium form] readily yields eq 1:
Figure 4. (A) Absorption spectra of malvin (0.1 mM) in the presence of
4-â-D-glucopyranosylferulic acid in a pH 2.5 phosphate buffer at 25 °C.
Copigment/pigment molar ratios: 0 (1), 10 (2), 20 (3), 30 (4), 40 (5). (B)
Plot of the visible absorbance at 530 nm as a function of copigment
concentration; experimental points (2) and theoretical curve (−) from a
curve fitting according to eq 1 (pKh set at 1.52).
Table 1. Copigmentation Binding Constants for the Different
Malvin
−
Copigment Couples Investigateda
-1
copigment
K (M
)
r 2
p-coumaric acid
4- -glucopyranosylcoumaric acid
caffeic acid
4- -glucopyranosylcaffeic acid
129 (
120 (
111 (
121 (
243 (
236 (
244 (
234 (
331 (
277 (
234 (
±
4)
4)
6)
5)
8)
9)
12)
12)
8)
14)
12)
0.998
0.997
0.996
0.998
0.992
0.999
0.997
0.998
0.999
0.997
0.998
±
±
±
±
±
±
±
±
±
±
â
-
D
â
-D
ferulic acid
4- -glucopyranosylferulic acid
â
-D
a Each experiment is duplicated. Values in parentheses are the standard
deviations of the curve-fitting procedure.
Equation 1 is used for the curve fitting of the A versus Lt
plots to obtain optimized values for K as the sole adjustable
parameter (Table 1). Overall, the glucosyl moiety of the
copigment has little impact on the stability of the copigmentation
complexes. A slight destabilization can be noted, especially for
ferulic acid, which can be attributed to the glucosyl moiety
hindering the pigment approach on one side of hydroxycinnamic
nucleus. The values of copigmentation binding constants are
close to the one reported in the literature for the malvin-
chlorogenic acid couple (31). However, they are ca. 1 order of
magnitude higher than those already reported with the copig-
ments caffeic acid and ferulic acid (32). However, in the quoted
work (32), a simplified theoretical treatment was used that
assumes the colored forms to be negligible with respect to the
colorless forms at the pH investigated. This assumption is
actually not valid at pH 2.5 so that a more general treatment
A0(1 + Kh/h)(1 + KLt)
A )
(1)
1 + Kh/h + KLt
In the absence of copigment, eq 1 simply becomes eq 2:
Ar0ef
A0 )
(2)
1 + Kh/h
Equation 2 is used for the determination of pKh at different
temperatures (25-45 °C). From a plot of ln Kh versus 1/T, we
deduce ∆Hh° ) 5.6 ((0.3) kJ/mol, ∆Sh° ) -11 ((1) J/K/mol.

7578 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Galland et al.
In this work, efficient chemical syntheses of O-aryl-â-D-
glucosides and 1-O-â-D-glucosylesters of hydroxycinnamic acids
are described. These naturally occurring polyphenols can be used
as standards for the identification and titration purposes in plants
and foods and for investigations of their bioavailability and
potential health effects. In particular, their glucosyl moiety could
allow a better intestinal absorption than the corresponding
aglycones. It is also noteworthy that 1-O-â-D-caffeoylglucose
retains a free catechol moiety that is critical for metal com-
plexation and a strong radical scavenging capacity. Finally,
although increasing their water solubility, the glucosyl moiety
does not significantly alter the capacity of the hydroxycinnamic
acid O-aryl-â-D-glucosides to interact with anthocyanins and
stabilize natural colors.
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−
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p-coumaric acid
∆
H 0 (kJ/mol)
∆
S 0 (J/K/mol)
r 2
−
−
−
−
−
−
32.1 (
±
±
±
±
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1.9)a
3.4)
4.7)
4.3)
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−
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−
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62.8 (
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0.990
0.990
0.994
0.993
4- -glucopyranosylcoumaric acid
caffeic acid
4- -glucopyranosylcaffeic acid
ferulic acid
4- -glucopyranosylferulic acid
â-
D
26.0 (
25.6 (
21.6 (
29.0 (
33.8 (
38 (
46 (
31 (
50.1 (
71 (
±
±
±
11)
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â-D
±
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D
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solely involving the planar polarizable polyphenolic nuclei.

Anthocyanin Copigmentation
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7579
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Received for review April 24, 2007. Revised manuscript received June
7, 2007. Accepted June 19, 2007.
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