Synthesis of a new pyranoanthocyanin dimer linked through a methyl-methine bridge

Article abstract of DOI:10.1016/j.tetlet.2011.03.125

Two new anthocyanin-derived compounds corresponding to the ethylpyranomalvidin-3-glucoside and the pyranomalvidin-3-glucoside dimer linked through a methyl-methine bridge were synthesized for the first time and their structure characterized by LC-DAD/MS a

Full text of DOI:10.1016/j.tetlet.2011.03.125

Tetrahedron Letters 52 (2011) 2957–2960
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of a new pyranoanthocyanin dimer linked through
a methyl-methine bridge
Joana Oliveira a, Nuno Mateus a, José E. Rodriguez-borges a, Eurico J. Cabrita b, Artur M. S. Silva c,
Victor de Freitas a,
⇑
a Centro de Investigação em Química, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal
b REQUIMTE/CQF, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
c Departamento de Química & QOPNA, Universidade de Aveiro, 3810-193 Aveiro, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new anthocyanin-derived compounds corresponding to the ethylpyranomalvidin-3-glucoside and
the pyranomalvidin-3-glucoside dimer linked through a methyl-methine bridge were synthesized for
the first time and their structure characterized by LC-DAD/MS and NMR spectroscopy. The latter was
obtained in a hydroalcoholic model solution through the reaction of the carboxypyranomalvidin-3-glu-
coside with ethylpyranomalvidin-3-glucoside and displays a blue/green color in solution.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 15 February 2011
Revised 21 March 2011
Accepted 23 March 2011
Available online 1 April 2011
Keywords:
Carboxypyranomalvidin-3-glucoside/vitisin A
Ethylpyranomalvidin-3-glucoside
Blue/green color
Pyranomalvidin-3-glucoside dimer
The color is an important feature and the first attribute to be
perceived in food and food derived products by the consumers.
Anthocyanins belong to the family of polyphenols that are respon-
sible for the color observed in many flowers and fruits and present
different colors from red to blue depending on the pH of the med-
ium.1,2 However, their color is only stable in aqueous solution at
low pH values (pH <3),1 conditions that are far from those com-
monly found in food matrixes. As such, Food Industry has been
searching for new natural compounds with different chromatic
properties presenting a higher stability toward the variation of
pH, temperature, light, etc.
In red wine, the color changes from red to red/orange during
aging due to the decrease on the anthocyanins that participate in
the formation of new anthocyanin-derived pigments having a wide
color range from yellow, red, purple, blue and turquoise.
These anthocyanin-derived compounds can be formed from the
condensation reaction between anthocyanins and catechins3–5 and
mainly from the reaction of anthocyanins with other non-polyphe-
nolic molecules such as acetaldehyde,6,7 acetoacetic acid,8 pyruvic
acid,9 hydroxycinnamic acids10,11 and other small molecules,12–14
leading to the formation of pyranoanthocyanin compounds.
Moreover, new families of compounds (portisins and pyranoanth-
ocyanin dimers) have been identified over the last years with
different chromatic and structural features (from violet/blue to
turquoise blue). All these pigments revealed higher color stability
with the increase of the pH of the medium.11,14–22 Bearing this,
the aim of this work was to synthesize a new pyranoanthocyanin
dimer 3 pigment derived from the reaction between two anthocy-
anin-derived compounds, carboxypyranoanthocyanin
1
and
ethylpyranoanthocyanin 2 (Scheme 1).
Firstly, ethylpyranomalvidin-3-glucoside 2 was synthesized
from the reaction between malvidin-3-glucoside and propionyl-
acetic acid23 according to the literature24 and mechanism pre-
sented in Scheme 2. The UV–vis spectrum of the newly formed
compound 2 (kmax = 478 nm) is similar to the one obtained and re-
ported in the literature for methylpyranomalvidin-3-glucoside8,21
and is expected to present similar physical–chemical features.
The full structural characterization of the ethylpyranomalvidin-3-
glucoside 2 was performed by 1H NMR (400.15 MHz) and 13C
NMR (100.12 MHz) on a Bruker–Avance 400 spectrometer at
293 K in DMSO-d6/TFA (9:1) (Table 1). The chemical shifts ob-
served for all the protons and carbons are in agreement with the
ones observed for methylpyranomalvidin-3-glucoside, except in
the substituent group linked to the carbon C-10 that in this case
is an ethyl (–CH2CH3) instead of a methyl (–CH3) group. The pro-
tons of the methylene (–CH2–) and the methyl (–CH3) groups ap-
pear on the spectrum as quartet (J = 7.1 Hz) and triplet
(J = 7.1 Hz) at 2.89 and 1.31 ppm, respectively. Their respective car-
bons were assigned through 1H–13C direct correlations.
⇑
Corresponding author. Tel.: +351 220402558; fax: +351 220402658.
E-mail address: vfreitas@fc.up.pt (V. de Freitas).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.03.125

2958
J. Oliveira et al. / Tetrahedron Letters 52 (2011) 2957–2960
OMe
OMe
OMe
OMe
OH
OMe
OH
OH
OH
+
+
HO
HO
HO
O
O
+
+
HO
O
O
OMe
OMe
OMe
+
OGlc
(2)
OGlc
OGlc
O-Glc
(1)
Carboxypyranomalvidin-3-glucoside
O
O
O
O
COOH
CH3
CH3
HOOC
Ethylpyranomalvidin-3-glucoside
Charge transfer complex
(π– πstacking)
OMe
3'
OH
2'
4'
+
B
8
1'
HO
8a
O
2
5' OMe
7
6
6'
A
C
3
4a
OGlc
5
4
OMe
D
O
9
5'
HO
4'
10
6'
1'
OGlc
B
9
MeO 3'
3
4
11
CH3
(3)
2
10
2'
D
C
4a
O
O
8a
8
5
A
6
7
OH
Pyranomalvidin-3-glucoside methyl-methine dimer
Scheme 1. Proposed chemical pathway for the formation of compound 3.
OMe
OH
Malvidin-3-glucoside
OMe
O
HO
OH
OMe
OMe
+
OH
HO
O
OGlc
OMe
O
HO
O
- H2O
OGlc
OMe
OH
CH3
OGlc
O
- HCO2H or
- CO2 + [O]
O
OMe
OMe
H
OH
OH
OH
OH
CH3
HO
O
+
HO
O
OMe
OMe
OGlc
OGlc
OH
O
propionylacetic acid
OH
OH
CH3
O
O
O
OH
Keto/enol
Ethylpyranomalvidin-3-glucoside
Scheme 2. Proposed mechanism for the formation of ethylpyranomalvidin-3-glucoside 2.
To synthesize the pyranoanthocyanin dimer linked through a
methyl-methine bridge, 10 mg of carboxypyranomalvidin-3-gluco-
side18 were incubated with 10 mg of ethylpyranomalvidin-3-glu-
coside23 (molar ratio of 1:1) in 10 mL of an aqueous solution of
20% ethanol at pH 4.0 (adjusted with a NaOH 0.01 M solution).
The mixture was left to react at 30 °C and the formation of new
pigments monitored by HPLC-DAD.25 After 5 days of reaction it
was possible to observe the appearance of a new chromatographic
peak 3 (Fig. 1) with two maximum absorption wavelengths at 697
and 637 nm (Fig. 2), thereby displaying a blue/green color.
of a molecular ion [M]+ at m/z 1059. Additionally, two major frag-
ment ions were detected at m/z 897 and 735 in the respective MS2
spectrum. The fragments correspond to the loss of one or two
glucose moieties, respectively. UV–vis and the LC–MS data are sim-
ilar to that published for a pyranoanthocyanin dimer.27 In order to
confirm this structure by NMR analysis, the compound was puri-
fied by TSK Toyopearl gel HW-40(S) column chromatography after
reaching its maximum formation (15 days) and further isolated by
semi-preparative HPLC. The yield of this reaction was of 15% being
obtained 3 mg of the pure compound.
The reaction mixture was analyzed by LC-DAD/MS26 and the
mass data recorded in the positive ion mode showed the presence
The identity of the pigment 3 (Scheme 1) was fully established
by 1H (600.13 MHz) and 13C NMR (150.62 MHz) using 1D and 2D

J. Oliveira et al. / Tetrahedron Letters 52 (2011) 2957–2960
2959
Table 1
1H and 13C chemical shifts of the new compound 2, determined in DMSO-d6/TFA (9:1)
247 nm
697 nm
Position
d
1Y ppm; J (Hz)
d
13C
HSQC
HMBC
Ethylpyranomalvidin moiety
637 nm
2
3
4
—
—
—
161.8
133.9
n.a.
—
—
—
H-20, 60B
H-9D
—
4a
5
6
7
8
8a
9
—
—
108.1
153.8
100.6
167.6
100.3
152.8
109.0
176.6
118.8
108.8
148.5
142.9
56.6
—
—
H-6A
—
H-8A
—
H-6, H-8
H-6
—
H-6, H-8
—
H-8
—
7.12; br s
—
7.32; br s
—
7.31; s
—
—
7.73; s
—
300
400
500
600
700
H-9D
—
wavelength (nm)
10
H-9, CH2, CH3
H-20, 60B
10
—
20,60
H-20, 60B
—
—
Figure 2. UV–vis spectra of compound 3 recorded from the HPLC-DAD.
30,50
H-20, 60B, OMe
40
—
—
H-20, 60B
30,50—OMe
–CH2–
–CH3
3.89; s
2.89; q, 7.1
1.31; t, 7.1
OMe
CH2
CH3
—
—
—
situated at 3.88 ppm was attributed to the protons of the methoxyl
groups present in positions 30 and 50 of the ring B (12H). The signal
situated at 2.20 ppm integrating 3 protons was attributed to the
protons of the –CH3 present in the methyl-methine linkage. The
anomeric proton of the glucose moiety H-100 was attributed to a
doublet (J = 7.2 Hz) situated at 4.77 ppm. All the protons of the glu-
cose molecules were situated at 3.08–3.49 ppm.
The assignment of the carbon resonances was possible using 2D
NMR techniques (gHSQC and gHMBC). Carbons C-20,60, C-9, C-6, C-
8, -OCH3, –CH3 and from glucose were assigned through their di-
rect correlation 1H-13C in the HSQC spectrum. Carbons C-10 and
C-11 were assigned, respectively, at 164.9 and 100.6 ppm from
their long-range correlation with the protons of the methyl group
of the methyl-methine linkage.
Concerning the mechanism of the reaction and attending to the
equilibrium forms of methylpyranomalvidin-3-glucoside in aque-
ous solutions reported recently at pH 4.0,21 this compound is pres-
ent in its pyranoflavylium cationic form in equilibrium with the
respective neutral quinoidal form. In a similar way the ethylpyr-
anomalvidin-3-glucoside is expected to be present in its pyranof-
lavylium cationic form under the reaction conditions (pH 4.0).
Thus, the condensation reaction of carboxypyranomalvidin-3-glu-
coside 1 with ethylpyranomalvidin-3-glucoside 2 to form com-
pound 3 may start with a charge-transfer reaction between the
28.1
10.9
Glucose moiety
100
4.68; d, 7.5
104.7
74.6
76.6
70.3
77.9
61.5
61.5
H-100
—
—
—
—
—
—
—
⁄
⁄
⁄
⁄
⁄
⁄
200
3.46;
3.26;
3.16;
3.05;
3.49;
3.28;
H-200
300
H-300
400
H-400
500
H-500
6a00
6b00
H-6a00, H-6b00
H-6a00, H-6b00
br s, broad singlet; s, singlet; d, doublet; t, triplet; q, quartet; ⁄, unresolved; n.a., not
assigned.
techniques (gCOSY, NOESY, gHMBC and gHSQC)28,29 (Table 2) in
DMSO-d6/TFA (9:1) on
CryoProbe.
a Bruker–Avance III 600 with a TCI
According to the proton and carbon chemical shifts (Table 2),
the double pyranoanthocyanin moieties are linked through a
methyl-methine bridge. Due to the symmetry plan present in the
dimer structure that passes through the methyl-methine carbon-
11, only four signals are expected to be found in the aromatic re-
gion of the proton spectrum. Indeed, protons H-6 and H-8 from
ring A were assigned to a singlet at 6.92 ppm integrating 4 protons
(4H). Proton H-9 from ring D was situated at 7.44 ppm (2H). Pro-
tons H-20,60 from ring B were situated at 7.57 ppm (4H). The signal
two pyranoflavylium moieties through
p–p stacking (Scheme 1)
and then ionic or radicalar reactions may occur, leading to the for-
1
80 0
700
2
600
500
400
300
200
100
478 nm
511 nm
3
696 nm
5
10
15
20
25
30
35
40
45
50
55
Retention Time
Figure 1. HPLC-DAD chromatogram of the synthesis of compound 3 from the reaction of carboxypyranomalvidin-3-glucoside 1 with ethylpyranomalvidin-3-glucoside 2
recorded at 480, 511 and 696 nm.

2960
J. Oliveira et al. / Tetrahedron Letters 52 (2011) 2957–2960
Table 2
8. He, J.; Santos-Buelga, C.; Silva, A. M. S.; Mateus, N.; de Freitas, V. J. Agric. Food
1H and 13C chemical shifts of the new compound 3, determined in DMSO-d6/TFA (9:1)
Chem. 2006, 54, 9598–9603.
9. Fulcrand, H.; dos Santos, P. J. C.; Sarni-Manchado, P.; Cheynier, V.; Favre-
Bonvin, J. J. Chem. Soc., Perkin Trans. 1 1996, 735–739.
Position
d
1Y ppm; J (Hz)
d
13C
HSQC
HMBC
10. Rentzsch, M.; Schwarz, M.; Winterhalter, P.; Hermosin-Gutierrez, I. J. Agric.
Food Chem. 2007, 55, 4883–4888.
Pyranomalvidin moieties
11. Schwarz, M.; Wabnitz, T. C.; Winterhalter, P. J. Agric. Food Chem. 2003, 51,
3682–3687.
12. Fulcrand, H.; Benabdeljalil, C.; Rigaud, J.; Cheynier, V.; Moutounet, M.
Phytochemistry 1998, 47, 1401–1407.
13. Hayasaka, Y.; Asenstorfer, R. E. J. Agric. Food Chem. 2002, 50, 756–761.
14. Schwarz, M.; Jerz, G.; Winterhalter, P. Vitis 2003, 42, 105–106.
15. Asenstorfer, R. E.; Jones, G. P. Tetrahedron 2007, 63, 4788–4792.
16. Cruz, L.; Petrov, V.; Teixeira, N.; Mateus, N.; Pina, F.; de Freitas, V. J. Phys. Chem.
B 2010, 114, 13232–13240.
17. Oliveira, J.; de Freitas, V.; Silva, A. M. S.; Mateus, N. J. Agric. Food Chem. 2007, 55,
6349–6356.
18. Oliveira, J.; Fernandes, V.; Miranda, C.; Santos-Buelga, C.; Silva, A.; de Freitas,
V.; Mateus, N. J. Agric. Food Chem. 2006, 54, 6894–6903.
19. Oliveira, J.; Mateus, N.; de Freitas, V. Tetrahedron Lett. 2011, accepted for
publication.
20. Oliveira, J.; Mateus, N.; Silva, A. M. S.; de Freitas, V. J. Phys. Chem. B 2009, 113,
11352–11358.
2C
3C
4C
4aC
5A
6A
7A
8A
8aA
9D
—
—
—
—
—
6.92; s
—
6.92; s
—
7.44; s
—
—
7.57; s
—
—
3.88; s
n.a.
—
—
—
—
—
H-6A
—
H-8A
—
H-9D
—
—
H-9D
H-9D
H-6, H-8
H-6
—
H-6, H-8
—
H-8
—
CH3
H-20, 60B
138.9
139.0
103.5
151.4
99.3
163.8
99.3
151.4
99.5
164.9
119.7
107.8
147.8
154.2
56.0
10D
10B
—
20,60B
30,50B
40B
H-20, 60B
—
—
H-20, 60B, OMe
H-20, 60B
—
—
OMe
30,50—OMe
21. Oliveira, J.; Petrov, V.; Parola, A. J.; Pina, F.; Azevedo, J.; Teixeira, N.; Bras, N. F.;
Fernandes, P. A.; Mateus, N.; Ramos, M. J.; de Freitas, V. J. Phys. Chem. B 2011.
doi:10.1021/jp110593c.
22. Oliveira, J.; Santos-Buelga, C.; Silva, A. M. S.; de Freitas, V.; Mateus, N. Anal.
Chim. Acta 2006, 563, 2–9.
Methyl-methine linkage
11
—
100.6
12.9
—
CH3
CH3
—
–CH3
2.20; s
Glucose moieties
23. Synthesis, purification, and structural characterization of the ethylpyranomalvidin-
3-glucoside: Propionylacetic acid was obtained from the hydrolysis of the ethyl
propionylacetate under very acid conditions (pH ꢀ0.5, HCl) for approximately
12 h. The obtained acid was further purified through extraction with ethyl
acetate. The organic fraction containing the acid was dried with anhydrous
sodium sulphate and filtered to remove the sodium sulphate. The ethyl acetate
was then eliminated from the organic fraction in a rotavaporator at 30 °C.
100 mg of malvidin-3-glucoside were incubated with 1.102 g of
propionylacetic acid (molar ratio of 1:50) in 100 mL of water at pH 2.60. The
reaction mixture was left to react at 30 °C and monitored the formation of new
compounds by HPLC-DAD. After 15 days, the reaction was stopped and the
main compound purified by column chromatography with TSK Toyopearl gel
HW-40(S). The fraction containing the compound was eluted from the gel with
an aqueous solution of 5% (v/v) methanol. The main compound was isolated by
semi-preparative HPLC and its structural features studied by LC–MS and NMR.
11 mg of the compound were obtained, which corresponds to a yield of around
10%. The full structural characterization of the ethylpyranomalvidin-3-
glucoside was performed by 1H NMR (400.15 MHz) and 13C NMR
(100.12 MHz) on a Bruker–Avance 400 spectrometer at 293 K in DMSO-d6/
TFA (9:1).
100
4.77; d, 7.2
3.45; t, 8.1
103.5
74.5
76.5
70.2
77.6
61.2
61.2
H-100
—
—
—
—
—
—
—
200
H-200
⁄
⁄
300
3.23;
H-30
400
3.11;
H-40
500
3.08⁄
H-50
⁄
⁄
6a00
6b00
3.26;
3.49;
H-6a00, 6b00
H-6a00, 6b00
s, singlet; d, doublet; t, triplet; ⁄, unresolved; n.a., not assigned.
mation of the pyranomalvidin-3-glucoside methyl-methine dimer
pigment. However, reaction involving methylpyranoanthocyanins
and ethylpyranoanthocyanins with other molecules such as carb-
oxypyranoanthocyanins through charge-transfer stacking still
need to be clarified.
24. Oliveira, J.; de Freitas, V.; Mateus, N. Tetrahedron Lett. 2009, 50, 3933–3935.
25. HPLC-DAD: All the reaction mixtures were monitored by HPLC-DAD analysis on
a Knauer K-1001 HPLC with a 250 ꢁ 4.6 mm i.d. reversed-phase C18 column
(Merck, Darmstadt). The detection was performed using a Knauer K-2800
diode array detector. For the synthesis of the carboxypyranomalvidin-3-
glucoside and ethylpyranomalvidin-3-glucoside the solvents used were A:
H2O/HCOOH (9:1) and B: HCOOH/H2O/CH3CN (1:6:3). For monitoring the
Acknowledgments
The authors thank Dr. Zélia Azevedo for the LC-DAD/MS analy-
sis and Dr. Mariana Andrade for the NMR analysis. The authors also
thank LabRMN at FCT-UNL and Rede Nacional de RMN for access to
the facilities. Rede Nacional de RMN is supported with funds from
FCT, Projecto de Re-equipamento Científico, Portugal. This research
was supported by a post-doctoral grant from FCT (Fundação para a
Ciência e a Tecnologia—Praxis BPD/65400/2009) and a grant from
FCT (Fundação para a Ciência e a Tecnologia—PTDC/AGR-ALI/
65503/2006, PTDC/QUI/67681/2006 and REDE/1517/RMN/2005)
all from Portugal and by FEDER funding.
reaction
between
carboxypyranomalvidin-3-glucoside
and
ethylpyranomalvidin-3-glucoside, the solvent
A
was the same described
previous and solvent B was HCOOH/H2O/CH3CN (0.5:19.5:80). For both cases,
the gradient consisted of 20–85% B for 70 min at a flow rate of 1.0 mL/min. The
column was washed with 100% B for 20 min and then stabilized with the initial
conditions for another 20 min. The conditions used for the semi-preparative
HPLC analysis were the same as just described except for the injection volume
which was 1 mL.
26. LC-DAD/MS: The reaction mixtures were analyzed by LC–MS on a Finnigan
Surveyor series liquid chromatograph, equipped with a 150 ꢁ 4.6 mm i.d.,
5 l
m LicroCARTÒ reversed-phase C18 column thermostatted at 25 °C. The
References and notes
mass detection was carried out by a Finnigan LCQ DECA XP MAX (Finnigan
Corp., San José, CA, USA) mass detector with an API (Atmospheric Pressure
Ionization) source of ionization and an ESI (ElectroSpray Ionization) interface.
The solvents used were A: H2O/TFA (99.9:0.1), and B: HCOOH/H2O/CH3CN
(0.5:19.5:80). The HPLC gradient used was the same reported above for the
HPLC analysis. The capillary voltage and temperature were 4 V and 325 °C,
respectively. Spectra were recorded in positive ion mode between m/z 300 and
1500. The mass spectrometer was programmed to do a series of three scans: a
full mass, a zoom scan of the most intense ion in the first scan, and a MS–MS of
the most intense ion using relative collision energy of 30 and 60 V.
1. Brouillard, R. Anthocyanins as Food Colors; Academic Press Inc.: New York, 1982.
pp 1–38.
2. Von Elbe, J. H.; Schwartz, S. J. Food Chemistry; Marcel Dekker Inc.: New York,
1996. pp 651–723.
3. Liao, H.; Cai, Y.; Haslam, E. J. Sci. Food Agric. 1992, 59, 299–305.
4. Remy, S.; Fulcrand, H.; Labarbe, B.; Cheynier, V.; Moutounet, M. J. Sci. Food Agric.
2000, 80, 745–751.
5. Santos-Buelga, C.; Bravo-Haro, S.; Rivas-Gonzalo, J. C. Z. Lebensm. Unters. Forsch.
1995, 201, 269–274.
27. Oliveira, J.; Azevedo, J.; Silva, A. M. S.; Teixeira, N.; Cruz, L.; Mateus, N.; de
Freitas, V. J. Agric. Food Chem. 2010, 58, 5154–5159.
28. Bax, A.; Subramanian, S. J. Magn. Reson. 1986, 67, 565–569.
6. Atanasova, V.; Fulcrand, H.; Le Guernevé, C.; Cheynier, V.; Moutounet, M.
Tetrahedron Lett. 2002, 43, 6151–6153.
7. Bakker, J.; Timberlake, C. F. J. Agric. Food Chem. 1997, 45, 35–43.
29. Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093–2094.