- Email: [email protected]
Influence of the structural features of amino-based pyranoanthocyanins on their acid-base equilibria in aqueous solutions
Accepted Manuscript Influence of the structural features of amino-based pyranoanthocyanins on their acidbase equilibria in aqueous solutions Joana Oliveira, Paula Araújo, Ana Fernandes, Natércia F. Brás, Nuno Mateus, Fernando Pina, Victor de Freitas PII:
S0143-7208(17)30073-6
DOI:
10.1016/j.dyepig.2017.03.005
Reference:
DYPI 5831
To appear in:
Dyes and Pigments
Received Date: 12 January 2017 Revised Date:
1 March 2017
Accepted Date: 2 March 2017
Please cite this article as: Oliveira J, Araújo P, Fernandes A, Brás NF, Mateus N, Pina F, de Freitas V, Influence of the structural features of amino-based pyranoanthocyanins on their acid-base equilibria in aqueous solutions, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.03.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Influence of the structural features of amino-based pyranoanthocyanins on their
2
acid-base equilibria in aqueous solutions.
3 Joana Oliveira1,2*, Paula Araújo1, Ana Fernandes1, Natércia F. Brás3, Nuno Mateus1,
5
Fernando Pina4, Victor de Freitas1 1
6
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REQUIMTE – Laboratório Associado para a Química Verde, Departamento de
Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo
8
Alegre, 687, 4169-007 Porto, Portugal, 2
ICETA – Instituto de Ciências, Tecnologias e Agroambiente da Universidade do Porto,
10
Praça Gomes Teixeira, Apartado 55142, 4051-401 Porto, Portugal, 3
11 12
REQUIMTE – UCIBIO, Departamento de Química e Bioquímica, Faculdade de
Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal, 4
13
REQUIMTE – Laboratório Associado para a Química Verde, Departamento de
Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516
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Monte de Caparica, Portugal.
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*
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Tel: +351.220402596
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Author to whom correspondence should be addressed, [email protected]
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REQUIMTE
22
ICETA
23 24
ABSTRACT
1
ACCEPTED MANUSCRIPT The equilibrium forms of three different
families of dimethylamino-based
26
pyranoanthocyanins (1, 2 and 3) were studied in aqueous solutions at different pH
27
values from 1 to 12 using UV-Visible spectroscopy. The forms present under those
28
conditions are strongly correlated to the pyranoanthocyanin structural features. The
29
increase of the electronic delocalization helps the protonation at the amino group. At
30
very acidic pH condition (pH<0) the protonation at the amino group is observed for the
31
three pigments, but under less acidic conditions (pH~1) it only occurs for pigment 3
32
(pKa1=2.4±0.1) and at a lesser extent for pigment 2 (pKa1=1.1±0.1). At the same time,
33
the increase of the electronic delocalization on the amino-based pigments also favors the
34
deprotonation at the hydroxyl group present at carbon C-7 yielding the neutral quinoidal
35
base (pKa2=2.7±0.1, pKa2=4.8±0.1 and pKa2=5.4±0.1 for pigment 3, 2 and 1,
36
respectively). For pigment 3, the maximum molar fraction obtained for the
37
pyranoflavylium cation form is ~ 0.4 due to the proximity of the two acid-base
38
constants (pKa1 and pKa2) which indicates that at the pH range 1-5 three forms of the
39
compound are present in equilibrium (pyranoflavylium dication, pyranoflavylium cation
40
and neutral quinoidal base). The second deprotonation at the 4′-OH was less affected by
41
the structural features of the pigment with the ionization constant situated at pKa3~9
42
(pKa3=9.5±0.1, pKa3= 8.9±0.1 and pKa3=9.8±0.1 for pigment 1, 2 and 3, respectively).
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Keywords: amino-derived pyranoanthocyanins; vinylene linkage; butadienylidene linkage; protonation; deprotonation; UV-Visible; Density functional theory (DFT). 43 44
1. Introduction
45
Colour is the first quality parameter perceived in many products such as foodstuffs and
46
beverages, cosmetics, fabrics and others. Therefore, colorants (natural or synthetic) play
2
ACCEPTED MANUSCRIPT a crucial role to add value to the final product. Anthocyanins are natural pigments which
48
colour in aqueous solution is pH-dependent [1] and taking into account that the
49
hydration equilibrium constant (pKh) of anthocyanins is between 2 and 3, in the
50
majority of food matrices it is expected that anthocyanins occur largely as colorless
51
hemiketals (> 70%) in equilibrium with other forms.
52
Over the years, different families of anthocyanin-derived pigments have been described
53
in the literature, the majority being identified in wine matrices during the ageing
54
process, namely anthocyanin-flavanols (linked directly or mediated by aldehydes) [2-4],
55
A- and B-type vitisins [3], methylpyranoanthocyanins [5], oxovitisins [6],
56
acetylpyranoanthocyanins [7], pyranoanthocyanin-flavanols [8], pyranoanthocyanin-
57
phenols
58
Pyranoanthocyanins, the main anthocyanin-derived compounds found in nature, present
59
a vast palette of colours ranging from yellow to turquoise blue [6, 13-16], which can
60
constitute a challenging research field since natural blue colored pigments are rare in
61
nature. Moreover, studies in aqueous solutions using UV-Visible and NMR techniques
62
showed that pyranoanthocyanins present a higher colour stability when compared to
63
their anthocyanin precursors which can be explained by the absence of hydration
64
reactions in those anthocyanin-derivatives [15-17]. On the other hand, it has been
65
reported that the presence of a dimethylamino group in a cinnamyl moiety of a
66
pyranoanthocyanin compound created a bathochromic shift of ~40 nm when compared
67
to a similar compound containing two hydroxyl groups [18]. In fact, the presence of
68
amino groups in synthetic flavylium compounds (or flavylia) has already shown to
69
create an important bathochromic displacement in the maximum wavelength of the
70
compounds leading to the formation of bluish molecules [19-21].
10],
portisins
[11,
12]
and
pyranoanthocyanin
dimers
[13].
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ACCEPTED MANUSCRIPT 71
Bearing this, three different bluish amino-derived pyranomalvidin-3-O-glucoside
72
pigments were synthesized from the reaction of malvidin-3-O-glucoside with 4-
73
(dimethylamino)-cinnamic acid (Pigment 1) [18], carboxypyranomalvidin-3-O-
74
glucoside
75
methylpyranomalvidin-3-O-glucoside with 4-(dimethylamino)-cinnaldehyde (Pigment
76
3) [23] and their network of equilibrium forms were studied in aqueous solutions at
77
different pH from 1 to 12 by UV-Visible spectroscopy. The chromatic feature of these
78
amino-derived pyranoanthocyanins brings promising expectations concerning their use
79
in Industry as colorants and dyes.
4-(dimethylamino)-cinnamic
acid
(Pigment
2)
[22]
and
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2. Material and Methods
82
2.1. Reagents
83
pH 1.00 (chloride acid/potassium chloride), 4.00 (sodium citrate) and 7.00 (sodium
84
phosphate) buffer solutions were obtained from Fluka (Madrid, Spain). A universal
85
buffer (1 L) of Theorell and Stenhagen [24] was prepared dissolving 2.25 mL of
86
phosphoric acid 85% (w/w), 7.00 g of monohydrated citric acid, 3.54 g of boric acid and
87
343 mL of a 1 M NaOH solution.
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2.2. Hemi-synthesis of amino based malvidin-3-O-glucoside pigments 1, 2 and 3
90
4-(Dimethylamino)-cinnamyl-pyranomalvidin-3-O-glucoside- (1) was obtained from
91
the reaction of malvidin-3-O-glucoside with 4-(Dimethylamino)-cinnamic acid
92
according to the procedure reported in the literature [18]. 4-(Dimethylamino)-cinnamyl-
93
10-vinylene-pyranomalvidin-3-O-glucoside (2) was synthesized from the reaction of the
94
carboxypyranomalvidin-3-O-glucoside with 4-(Dimethylamino)-cinnamic acid as
95
described
previously
[22].
4-(Dimethylamino)-cinnamyl-10-butadienylidene-
4
ACCEPTED MANUSCRIPT 96
pyranomalvidin-3-O-glucoside (3) pigment was obtained from the reaction of the
97
methylpyranomalvidin-3-O-glucoside with
98
reported elsewhere [23]. The purity of all pigments was confirmed by NMR.
4-(Dimethylamino)-cinnamaldehyde as
99 2.3. Titration of pigments 1, 2 and 3 by UV-Visible spectroscopy
101
Stock solutions (0.24 mM) were prepared for pigments 1, 2 and 3 in an aqueous solution
102
of 45% (v/v) ethanol (to obtain a final concentration of 15% (v/v) ethanol) 0.1 M HCl. 1
103
mL of a 0.1 M NaOH solution was added to a 10x10 mm quartz cell, 1 mL of Theorell
104
and Stenhagen universal buffer solution at pH~1 and 1 mL of the stock solution (the
105
pigment final concentration was 0.08 mM). The titrations of the pigments were
106
performed until pH~12 by the addition of small volumes (1-5 µL) of base (1 M or 10 M
107
NaOH). For each pigment, the first spectrum (250-900 nm) was run immediately after
108
the addition of the stock solution and shaking the cell. Successive spectra were recorded
109
instantly after the addition of the base in a Thermo Scientific Evolution Array UV-
110
Visible spectrophotometer at 25ºC. The obtained spectra were adjusted for the final
111
volume in each point. The pH values of the initial solution and after the addition of the
112
base were measured in a WTW pH 320 (Weilheim, Germany) with a CRISON 5209
113
combined glass electrode of 3 mm diameter (Barcelona, Spain). The pH meter was
114
calibrated with pH 1 and 4 buffer solutions for pH values below 2.5 and, pH 4 and 7
115
buffer solutions for pH values above 2.5. The fittings for pKa’s determination were
116
carried out using the Solver program from Microsoft Excel.
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117 118
2.4. Theoretical calculation for UV-visible properties
119
Density functional theory (DFT) calculations with the hybrid functional B3P86 and the
120
basis set 6-31+G(d,p) were used to optimize the geometry of the twelve
5
ACCEPTED MANUSCRIPT pyranoanthocyanin equilibrium forms (dicationic, cationic, neutral quinoidal and
122
anionic quinoidal forms for each pigment). This functional and basis set were
123
specifically chosen due to its good ability to calculate thermodynamic and UV-visible
124
absorption properties for polyphenols [25-27]. Frequency calculations were carried out
125
at the same level of theory in order to confirm the absence of imaginary frequencies in
126
the ground state. Subsequently, Time Dependent (TD) DFT single-point calculations
127
with the same level of theory were applied to determine the excited singlet state
128
energies, and subsequently, the allowed vertical π → π∗ electronic excitation energies
129
were obtained. These provide the absorption energies in the UV-visible range with the
130
contribution of all one-electron transitions and their oscillator strength.
131
The TD-DFT calculations were performed in vacuum and in solvent. For continuum
132
solvent calculations, the integral equation formalism Polarizable Continuum Model
133
(IEFPCM) method with a dielectric constant (ε) of 80 was used. This value of ε allows
134
to evaluate the long-range effect of an aqueous environment. All calculations were
135
carried out with the Gaussian09 software package [28].
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136
3. Results and discussion
138
The equilibrium forms of three families of amino-based pyranoanthocyanins (Figure 1)
139
were evaluated in aqueous solutions at different pH values from 1 to 12 by UV-Visible
140
spectroscopy.
141
pyranomalvidin-3-O-glucoside moiety linked to a 4-(dimethylamino)-cinnamyl group.
142
The structural differences between the three pigments are in the type of linkage
143
preforming the connection between both moieties. In pigment 1 the moieties are linked
144
directly through a C – C bond. In the case of pigment 2 the connection is made through
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The
studied
compounds
are
similar
molecules
containing
a
6
ACCEPTED MANUSCRIPT a vinylene linkage and in pigment 3 through a butadienylidene one. These structural
146
features confer to the pigments different colours and stabilities in aqueous solutions.
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Figure 1 – Structure of the three amino-derived pyranoanthocyanin pigments in their
150
pyranoflavylium cation form.
151
3.1. Equilibrium forms in aqueous solutions at different pH values
153
3.1.1. 4-(dimethylamino)-cinnamyl-10-pyranomalvidin-3-O-glucoside (1)
154
The maximum absorption wavelength for pigment 1, in aqueous solution at acidic pH
155
value (pH 1.99) is 557 nm and the compound displays a violet colour. With the increase
156
of the pH value until 6.13 a decrease in the absorbance at the maximum wavelength is
157
observed together with a hypsochromic shift and the appearance of a shoulder around
158
500 nm that increases with pH (Figure 2A). This was attributed to the equilibrium
159
between the pyranoflavylium cation form and the neutral quinoidal base. This
160
equilibrium is confirmed by the presence of an inflection point in the titration curve of
161
the absorbance at 557 nm versus the pH in this pH range (Figure 3A). Although it is not
162
usual to observe quinoidal bases of pyranoanthocyanins that absorb close to 500 nm,
163
this behavior has already been reported in the literature by Schwarz and Winterhalter for
164
this compound [18], by Oliveira et al. for similar compounds [12, 14] and more recently
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ACCEPTED MANUSCRIPT by Vallverdú-Queralt et al. for two pyranoanthocyanins [29]. For higher pH values up
166
to ~12, it is observed the increase of the absorbance at 500 nm and the appearance of a
167
shoulder at 600 nm (Figure 2B). This trend can be due to the equilibrium between the
168
pyranoflavylium neutral quinoidal form and the respective anionic form that is
169
supported by the second inflection point observed in the titration curve in this pH range
170
(Figure 3A).
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1.8
pH 1.99 1.6
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1.4
pH 6.13
1 0.8 0.6 0.4
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Absorbance
1.2
A
0.2 0 350
450
550
λ (nm)
650
750
850
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1.2
1
pH 11.85
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Absorbance
0.8
pH 7.59
0.6
AC C
0.4
B
0.2
0
250
171
350
450
550
650
750
850
λ (nm)
172
Figure 2 – UV-Visible spectra of pigment 1 in aqueous solution at different pH values A: from
173
1.99 to 6.13; B: from 7.59 to 11.85.
8
ACCEPTED MANUSCRIPT 1.8 1.6
A
Absorbance
1.4 1.2
pKa2 = 5.4±0.1
1.0 0.8
557 nm 470 nm
pKa3 = 9.5±0.1
0.6
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0.4 0.2 0.0 5
7 pH
B
pKa1 = 1.1±0.1
pKa2 = 4.8±0.1
420 nm
3.00
5.00
7.00 pH
11.00
13.00
C
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pKa1 = 2.4±0.1
9.00
510 nm
pKa3 = 9.8±0.1
pKa2 = 2.7±0.1
1 0.5
AC C
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665 nm
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Absorbance
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520 nm
pKa3 = 8.9±0.1
2
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11
551 nm
2.5
1.5
9
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1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1.00
3
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1
3.00
5.00
7.00 pH
9.00
11.00
13.00
175
Figure 3 – Titration curves of the absorbance at different wavelengths versus pH and respective
176
pKa ’s at T=298 K at pH 1–12 for A: for pigment 1; B: for pigment 2; C: for pigment 3.
177 178
3.1.2. 4-(dimethylamino)-cinnamyl-10-vinylene-pyranomalvidin-3-O-glucoside (2)
179
In the case of pigment 2, the UV-Visible spectrum in acid conditions (pH 1.44) showed
180
a maximum absorption wavelength at 520 nm (Figure 4A). Under these conditions this 9
ACCEPTED MANUSCRIPT compound presents a red colour. With the increase of the pH up to 2.58 a decrease in
182
the absorbance at this wavelength is observed concomitantly with an increase at 633 nm
183
(Figure 4A). The first assumption that was made was that this would correspond to the
184
equilibrium between the pyranoflavylium cation form and the respective neutral
185
quinoidal form. However, the spectrum of the same compound determined at very low
186
pH values (pH=0, -0.5 and -0.7) showed the disappearance of the shoulder at 633 nm
187
and at the same time the increase of the absorbance at 520 nm (Figure 4A). The spectra
188
obtained for the solutions in very acidic conditions is similar to the one reported for the
189
pigment at pH 1.44. This indicates that the equilibrium forms that should be present at
190
these pH values are the protonated form (at the amino group) of the compound
191
(dication) in equilibrium with the respective pyranoflavylium cation form. In fact, the
192
protonation of amino groups has already been reported in the literature for synthetic
193
flavyliums [19, 20]. The detection of the pyranoflavylium dication form of pigment 2 by
194
ESI-MS was not possible to observe due to its low pKa1 value making difficult its
195
detection in the mass spectrum under the conditions used, contrarily to pigment 3 as
196
discussed below. The protonation of the amino group leads to an interruption in the
197
electronic delocalization of the molecule, explaining why although pigment 2 has higher
198
electronic delocalization than pigment 1 it presents an unexpected red colour in acidic
199
conditions.
200
With the increase of the pH of the solution for values up to 8.20 a reverse trend is
201
observed, with a decrease in the absorbance at 633 nm at the same time as the increase
202
of the absorbance at ~520 nm (Figure 4B). This corresponds to the equilibrium between
203
the pyranoflavylium cation form and the respective neutral quinoidal form. The titration
204
curve of the absorbance at 420, 520 and 551 nm presented in Figure 3A shows an
205
inflection point corresponding to this equilibrium. Moreover, at the pH range 7-12 is
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10
ACCEPTED MANUSCRIPT 206
observed the increase of the absorbance at ~520 nm and the equilibrium between the
207
neutral and the anionic quinoidal forms is observed in a similar manner as described for
208
pigment 1. In this pH range it is also detected an inflection point in the titration curves
209
(Figure 3A) confirming the postulated equilibrium.
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0.6
0.5 pH - 0.7 pH - 0.5
pH 2.58
pH 0
0.3
0.2
A
pH 1.44
0.1
0 350
450
550 λ (nm)
1.6
650
pH 2.71
1.4
1.2
pH 8.20
0.8
850
TE D
Absorbance
1
750
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250
0.6
0.4
0 250
350
EP
0.2
450
550
650
SC
Aborbance
0.4
750
B 850
λ (nm)
AC C
1.8
1.6
pH 11.77
1.4
Absorbance
1.2
1
pH 7.16
0.8
0.6
0.4
C
0.2
0 250
210
350
450
550
650
750
850
λ (nm)
11
ACCEPTED MANUSCRIPT 211
Figure 4 – UV-Visible spectra of pigment 2 in aqueous solution at different pH values A: from -
212
0.7 to 2.44; B: from 2.71 to 8.20; C: at 7.16 to 11.77.
213 3.1.3. 4-(dimethylamino)-cinnamyl-10-butadienylidene-pyranomalvidin-3-O-glucoside
215
(3)
216
For pigment 3, the UV-Visible spectra recorded under acidic conditions (pH 1.60)
217
showed a maximum absorption wavelength at 535 nm (Figure 5A). Since this pigment
218
presents higher electronic delocalization than pigments 1 and 2 it was expected to
219
present a more bluish colour. However, in acid conditions the pigment presents a red
220
colour and is hence thought to be present mainly in its dication pyranoflavylium form,
221
similarly to pigment 2 but in a greater extent. Moreover, the spectrum at pH 1.60 is very
222
similar to the one observed at very acidic conditions (pH 0, -0.5 and -0.7) (Figure 5A).
223
In fact, the presence of the dicationic form of this compound was already confirmed by
224
ESI-MS with m/z=m/2=344.67 in the positive ion mode [23]. With the increase of the
225
pH value up to 2.65 it is observed the decrease in the absorbance at this maximum
226
absorption wavelength together with the increase at 665 nm (Figure 5A). This was
227
attributed to the equilibrium between the pyranoflavylium dication and the cation forms.
228
For pH values between 2.67 and 5.68 a third absorption wavelength at 588 nm appears
229
and decreases in intensity with the increase of the pH (Figure 5B). This indicates that at
230
this pH range additionally to the pyranoflavylium dication and cation forms it is also
231
observed the presence of neutral quinoidal form. This is confirmed by the two inflexion
232
points observed in the titration curve of the absorbance at 510 and 665 nm versus the
233
pH (Figure 5B) at this pH range.
234
Contrarily to catechyl-pyranomalvidin-3-O-glucoside and guaiacyl-pyranomalvidin-3-
235
O-glucoside pigments that were reported by Vallverdú-Queralt et al. to be prone to
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12
ACCEPTED MANUSCRIPT aggregation above pH 4 when the neutral quinoidal base is mainly present [29], pigment
237
3 does not seem to aggregate at the concentration used in this study. In fact, the shape of
238
the spectra obtained for the pigment at pH 6.5 (where the neutral quinoidal base is
239
present) at different molar concentrations from 0.057 to 0.143 mM (Figure 1 –
240
Supplementary material) is similar. Moreover, by normalizing the spectra obtained by
241
the division of the absorbance at each wavelength by the molar concentration it is
242
possible to observe an almost complete juxtaposition of the spectra which also
243
corroborates with the absence of aggregation (Figure 2 – Supplementary material). In
244
addition, in Figure 3 (Supplementary material) it is also possible to observe only a
245
small change in the absorption spectra of pigment 3 with time (0, 2179 and 7185 s) at
246
pH 6.42.
247
For higher pH values up to 11.53, the absorbance at 535 nm increases along with the
248
decrease at 588 and 665 nm (Figure 5C). This tendency is due to the equilibrium
249
between the neutral and the anionic quinoidal forms.
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13
ACCEPTED MANUSCRIPT 1 0.9 0.8 pH -0.7 pH -0.5 pH 0
0.6 0.5 0.4
A
pH 2.65
0.3 0.2
pH 1.60
0.1
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Absorbance
0.7
0 250
350
450
550
650
750
850
λ (nm) 2
pH 2.41
SC
pH 2.67
pH 5.87 1
B
0.5
0 250
350
450
550
650
λ (nm) pH 11.53 2
850
pH 5.87
1
0 250
350
EP
0.5
450
550
650
750
C 850
λ (nm)
AC C
250
750
TE D
Absorbance
1.5
M AN U
Absorbance
1.5
251
Figure 5 – UV-Visible spectra of pigment 3 in aqueous solution at different pH values A: from -
252
0.7 to 2.65; B: from 2.41 to 5.87; C: from 5.87 to 11.53.
253 254
3.2. TD-DFT calculations
255
The UV-visible absorption spectra and the Molecular Orbitals (MO) correlation
256
diagrams for the four forms [dicationic (A), cationic (B), neutral (C) and anionic (D)] of
257
pigments 1, 2 and 3 were also determined by TD-DFT calculations (Figures 4 to 7 in
14
ACCEPTED MANUSCRIPT Supplementary material). The dicationic form of pigment 1 was predicted using TD-
259
DFT calculation as corresponding to the first excited state S0 → S1 (at 504.73 nm),
260
which is essentially related to the HOMO → LUMO electronic transition. Moreover,
261
the experimental large band at 557 nm (Figure 2A) exhibited by the cationic form of
262
pigment 1 which is predicted theoretically as the second excited state (S0 → S2) at
263
446.74 nm (Figure 5 in Supplementary material). This corresponds to the HOMO-1 →
264
LUMO electronic transition, in which both MO are being delocalized along the
265
extended conjugated path of the dimethylamino-cinnamyl-ring. Although the LUMO is
266
almost similar for dicationic and cationic forms, the first deprotonation induces a
267
stabilization of the HOMO and HOMO-1 due to the large delocalization occurred in the
268
dimethylamino-cinnamyl -ring, and subsequently decreases the energy gap and results
269
in a slight bathochromic shift. Concerning the neutral and anionic forms, the
270
experimental large band at 500 nm and shoulder at 600 nm are theoretically predicted
271
by the first excited states at 468.77 nm and 569.49 nm, respectively, which still ascribe
272
to the HOMO → LUMO electronic transition. The HOMO of neutral form is mainly
273
located at A-ring and dimethylamino-cinnamyl -ring, while in the anionic form it was
274
displaced from the dimethylamino-cinnamyl-ring to the B-ring.
275
The bands of dicationic and cationic forms of pigment 2 predicted by the TD-DFT
276
calculations correspond to the first excited state S0 → S1 (at 468.48 nm and 465.54 nm,
277
respectively) that is defined by the HOMO → LUMO electronic transition. As observed
278
in pigment 1, the LUMO is almost identical for both forms, whereas the HOMO is
279
severely displaced toward the amino-ring, which dramatically decreases the overlap
280
with the LUMO. This strong concentration of HOMO around the dimethylamino-
281
cinnamyl-ring (see Figure 6 of Supplementary material) occurs due to the higher
282
delocalization expected on this compound, in comparison with pigment 1. In relation to
AC C
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SC
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258
15
ACCEPTED MANUSCRIPT the neutral form, the TD-DFT predicts the first excited state at 481.03 nm that is
284
attributed to the HOMO → LUMO electronic transition, and both MO are mainly
285
concentrated in the A-ring. For the anionic form, the first excited state (corresponding to
286
the HOMO → LUMO electronic transition) was predicted at 540.36 nm. The wider
287
distribution of HOMO-1 than HOMO around the A-ring and B-ring is related to the
288
higher delocalization verified in this form, which justifies the higher wavelength and the
289
expectation of its bluish colour.
290
The theoretical UV-visible absorption spectra predicted for pigment 3 are very similar
291
to the ones observed for pigment 2. The dicationic, cationic, neutral and anionic have
292
first excited states (HOMO → LUMO electronic transition) at 465.44 nm, 464.54 nm,
293
480.40 nm and 538.36 nm, respectively. However, the cationic form of pigment 3 has
294
both HOMO and HOMO-1 mostly located along the amino group (Figure 7 of
295
Supplementary material), which indicates that the second excited state associated to the
296
HOMO-1 → LUMO electronic transition is also involved in the bluer colour of cationic
297
form than dicationic form. For anionic form, the HOMO → LUMO electronic transition
298
predicted at 538.36 nm (corresponding to the experimental band at 535 nm) is related to
299
the delocalization verified around the A-ring and B-ring.
300
Theoretical and experimental spectra do not overlap completely, especially those
301
concerning the dicationic pyranoflavylium forms and the anionic quinoidal forms.
302
However, it is not the first time that unusual chromatic features have been reported in
303
the literature for other pyranoanthocyanins, like the colour change from violet to blue
304
with the decrease of the temperature from 25ºC to -10ºC for aqueous solutions of
305
pyranoanthocyanin-vinyl-cinnamyl (Portisin B) pigments.
306
chemical change was explained to be due to electronic and vibrational effects [30].
307
Moreover, the hypsochromic shift observed in the maximum absorption wavelength for
AC C
EP
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SC
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283
This reversible physical-
16
ACCEPTED MANUSCRIPT pyranoanthocyanins in general (e.g. A-type and B-type vitisins) when compared to the
309
anthocyanin counterpart although they present a higher electronic delocalization was
310
explained using quantum theoretical studies by differences in the planarity of the
311
ubiquitous B ring in this kind of pigments [31]. The optimized geometry of quinoidal
312
anionic forms have a lesser planarity of the B-ring than the other forms (data not shown)
313
and that could explain the hypsochromic shift observed in the maximum absorption
314
wavelength for these equilibrium forms in aqueous solutions. In addition, the dicationic
315
pyranoflavylium forms also revealed a distortion from the planarity of the
316
dimethylaminocinnamyl-ring that should be due to the presence of the proton at the
317
dimethylamino group. Here, we only determined the UV-Vis spectrum for one
318
optimized conformation for each form of the three pigments. More precise
319
measurements (with a structural conformational study to determine the most
320
energetically stable conformation of each compound, and then calculate their UV-Vis
321
spectra or the average wavelength values of all conformations) could be required to
322
obtain more reliable data. However, they would represent a highly demanding approach
323
(in terms of computation time) considering the high number of forms and compounds,
324
as well as the large number of rotatable bond present in each molecule. On the other
325
hand, the DFT has an error associated, in particular the density functional B3P86 has a
326
deviation in the determination of maximum wavelength values of natural molecules
327
between 11 nm [25] and 24 nm [32]. Although this error is smaller than the one
328
obtained with other density functionals, it may justify, for example the discrepancy
329
occurred in the wavelength maximum values of dicationic and cationic forms of
330
pigments 2 and 3 (exp: cation > dication and teor: cation ≈ dication). Overall, a
331
combination of these effects may be responsible for the differences observed for some
332
theoretical and experimental spectra.
AC C
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308
17
ACCEPTED MANUSCRIPT 333 334
3.3. Structural features versus acid-base equilibria
335
Based
336
pyranoanthocyanins at different pH values from 1 to 12 it was possible to determine the
337
type of equilibria that were taking place in aqueous solutions for each pigment. In
338
general, pyranoanthocyanins can undergo two deprotonations at 7-OH from ring A and
339
at 4′-OH from ring B [15, 33]. In the case of pigments containing amino groups, an
340
additional protonation at this moiety can occur at acidic conditions [19, 20]. However,
341
in this study, it was demonstrated that the presence of an amino group and its distance to
342
the pyranoanthocyanin moiety has a great influence on the acid-base equilibria in
343
aqueous solutions of the pigments. According to the results discussed previously, in
344
aqueous solutions from pH 1 to 12, pigment 1 is present in three equilibrium forms, the
345
pyranoflavylium, the neutral and anionic quinoidal forms. In the case of pigments 2 and
346
3, the pyranoflavylium dication form is also present additionally to the other three
347
(Figure 6). After defining the particular equilibria forms for each pigment, the acid-base
348
ionization constants were determined, plotting the absorbance at selected wavelengths
349
as a function of the pH of the solution (Figure 4). The fittings for determination of the
350
pKa values were carried out using the Solver program from Microsoft Excel and are
351
presented in Table 1.
the
UV-Visible
spectra
obtained
for
the
three
amino-derived
AC C
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on
18
ACCEPTED MANUSCRIPT OCH3
OCH3
A
OH
OH
HO
HO
O
O OCH3
OCH3 O HO
OH OH
O
OH
O HO
OH
O
OH
O
OH
O
pKa1
H
H3C
CH3
CH3
RI PT
N
N H3C
H+
+ n = 0, 1, 2
n = 0, 1, 2
OCH3
OCH3
OH
OH O HO
SC
B
O
OCH3
O OCH3 OH
O
OH
O
O
O
M AN U
O HO
OH
O HO
OH
OH OH
+
H+
+
H+
pKa2
n = 0, 1, 2
n = 0, 1, 2
N
H3C
N CH 3 OCH3
C
TE D
H3C
CH3
OCH3
OH O
O
O O
O
OCH3 O HO
O
O HO
OH
OH
EP
O
OCH3
OH
pKa3
AC C
n = 0, 1, 2
N
H3C
CH3
O
OH OH
O
n = 0, 1, 2
352 353
OH
N H3C
CH3
354
Figure 6 – General representation of the proton transfer equilibria of the amino-derived
355
pyranoanthocyanins 1 (n=0), 2 (n=1) and 3 (n=2) in aqueous solutions at pH between 1 and 12. )
356 357
19
ACCEPTED MANUSCRIPT 358
Table 1 – pK a values of the three amino-derived pyranoanthocyanins determined by UV-Visible.
359
E, λ max determined in ethanol 0.01% HCl; λ max of AH 2 2+, AH + , A and A- determined from the
360
experimental data (s, indicates a shoulder). λmax (nm)
pKa
E
AH22+
AH+
A
A-
1
562
n.d.
557
512
509
632
519
633
623, 519
543, 612
(s)
(s)
554
531, 612
2
668
531
625
<1
pKa2
pKa3
5.4±0.1
9.5±0.1
1.1±0.1 4.8±0.1
8.9±0.1
2.4±0.1 2.7±0.1
9.8±0.1
SC
3
pKa1
RI PT
Pigment
(s)
M AN U
361
The acid-base constants presented in Table 1 indicate that the protonation at the amino-
363
group and the deprotonation at the hydroxyl group present in carbon C-7 from ring A
364
are the most affected equilibria by the pigments structural features. Pyranoanthocyanins
365
linked directly to the 4-(dimethylamino)-cinnamyl (pigment 1) are more resistant to the
366
protonation (pKa1<1) at the amino group than pyranoanthocyanins linked to the 4-
367
(dimethylamino)-cinnamyl by a vinylene group (pigment 2) (pKa1=1.1±0.1) or by a
368
butadienylidene one (pigment 3) (pKa1=2.4±0.1). This latter was the most susceptible to
369
the protonation reaction presenting a lower ionization constant. In a similar manner, the
370
deprotonation reaction at 7-OH from the ring A of the pyranoflavylium cation to yield
371
the neutral quinoidal form is correlated to the pigment electronic delocalization. It
372
occurs more easily for pigment 3 (pKa2=2.7±0.1) with higher electronic delocalization,
373
than for pigment 2 (pKa1=4.8±0.1) and pigment 1 (pKa2=5.4±0.1). On the other hand,
374
the second deprotonation at 4′-OH is the less affected acid-base equilibrium by the
375
structural features of the molecule (pigments 1, 2 and 3 with pKa3=9.5±0.1,
376
pKa3=8.9±0.1 and pKa3=9.8±0.1). This tendency can be easily observed in the molar
AC C
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362
20
ACCEPTED MANUSCRIPT fraction of the network of equilibrium forms for the three pigments studied presented in
378
Figure 7.
379
It seems that the presence of a butadienylidene linkage (pigment 3) in a
380
pyranoanthocyanin compound stabilizes the deprotonated forms of the compound when
381
compared with similar compounds presenting a vinylene group (pigment 2). This has
382
already been observed in a minor extent for similar pigments containing a sinapyl
383
moiety linked to pyranoanthocyanins [33].
AC C
EP
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M AN U
SC
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377
21
ACCEPTED MANUSCRIPT 1.0
AH+
0.9
A-
A
Molar fraction
0.8 0.7 0.6 0.5 0.4 0.3
A
0.1 0.0 1
3
5
7
9
11
pH 1.0 0.9
A-
A
AH+
SC
0.7 0.6 0.5 0.4 0.3
AH22+
0.2
B
0.1 0.0 1.00
3.00
5.00
7.00
pH 1.0
AH22+
9.00
0.6
AH+
11.00
A-
EP
0.4
TE D
A
0.8
Molar fraction
M AN U
Molar fraction
0.8
RI PT
0.2
0.2
C
AC C
0.0
1.00
3.00
5.00
7.00
9.00
11.00
pH
384 385
Figure 7 – Molar fraction distribution diagram for pigment A: 1; B: 2 and C: 3 as a function of the
386
pH obtained from the UV-Visible determination.
387 388
4. Conclusions
389
The structural differences concerning the three pyranomalvidin-3-O-glucoside
390
derivatives is in the type of linkage performing the connection between the pyran 22
ACCEPTED MANUSCRIPT moiety and the dimethylaminophenyl unit yielding pigments with different structural
392
features and electronic delocalization. It was observed by UV-Visible spectroscopy that
393
the forms present in aqueous solutions at different pH values from 1 to 12 are strongly
394
correlated to the pyranoanthocyanin structural features. It seems that the increase of the
395
electronic delocalization helps the protonation at the amino group since it was observed
396
that the protonation was favored for pigment 3, the one that presents a higher electronic
397
delocalization and then for pigment 2. This kind of equilibrium was not observed for
398
pigment 1 in the conditions studied.
399
With respect to the color stability and concerning the possible applications of these
400
pigments as colorants, it appears that pigment 3 is the less viable since at the pH of most
401
food matrices (pH 2-5) this pigment is present in three different forms in equilibrium
402
with the pyranoflavylium cation form (blue color) accounting for only 40% (Figure
403
7C). The most stable compound at those pH values is pigment 1 (Figure 7A) although
404
the color presented is not the most interesting one.
405
TE D
M AN U
SC
RI PT
391
5. Literature cited
407
[1] Brouillard R, Dubois J-E. Mechanism of the structural transformations of
408
anthocyanins in acidic media. J Am Chem Soc. 1977;99(5):1359-64.
409
[2] Pissarra J, Mateus N, Rivas-Gonzalo J, Buelga CS, de Freitas V. Reaction between
410
malvidin 3-glucoside and (+)-catechin in model solutions containing different
411
aldehydes. J Food Sci. 2003;68(2):476-81.
412
[3] Salas E, Atanasova V, Poncet-Legrand C, Meudec E, Mazauric JP, Cheynier V.
413
Demonstration of the occurrence of flavanol-anthocyanin adducts in wine and in model
414
solutions. Anal Chim Acta. 2004;513(1):325-32.
AC C
EP
406
23
ACCEPTED MANUSCRIPT [4] Salas E, Le Guerneve C, Fulcrand H, Poncet-Legrand C, Cheynier W. Structure
416
determination and colour properties of a new directly linked flavanol-anthocyanin
417
dimer. Tetrahedron Lett. 2004;45(47):8725-9.
418
[5] He J, Santos-Buelga C, Silva AMS, Mateus N, De Freitas V. Isolation and structural
419
characterization of new anthocyanin-derived yellow pigments in aged red wines. J Agric
420
Food Chem. 2006;54(25):9598-603.
421
[6] He J, Oliveira J, Silva AMS, Mateus N, De Freitas V. Oxovitisins: a new class of
422
neutral pyranone-anthocyanin derivatives in red wines. J Agric Food Chem.
423
2010;58(15):8814-9.
424
[7] Gomez-Alonso S, Blanco-Vega D, Victoria Gomez M, Hermosin-Gutierrez I.
425
Synthesis, isolation, structure elucidation, and color properties of 10-acetyl-
426
pyranoanthocyanins. J Agric Food Chem. 2012;60(49):12210-23.
427
[8] He J, Santos-Buelga C, Mateus N, de Freitas V. Isolation and quantification of
428
oligomeric pyranoanthocyanin-flavanol pigments from red wines by combination of
429
column chromatographic techniques. Journal of Chromatography A. 2006;1134(1-
430
2):215-25.
431
[9] Schwarz M, Jerz G, Winterhalter P. Isolation and structure of pinotin A, a new
432
anthocyanin derivative from Pinotage wine. Vitis. 2003;42(2):105-6.
433
[10] Schwarz M, Wabnitz TC, Winterhalter P. Pathway leading to the formation of
434
anthocyanin−vinylphenol adducts and related pigments in red wines. J Agric Food
435
Chem. 2003;51(12):3682-7.
436
[11] Mateus N, Silva AMS, Rivas-Gonzalo JC, Santos-Buelga C, De Freitas V. A new
437
class of blue anthocyanin-derived pigments isolated from red wines. J Agric Food
438
Chem. 2003;51(7):1919-23.
AC C
EP
TE D
M AN U
SC
RI PT
415
24
ACCEPTED MANUSCRIPT [12] Oliveira J, de Freitas V, Silva AMS, Mateus N. Reaction between
440
hydroxycinnamic acids and anthocyanin-pyruvic acid adducts yielding new portisins. J
441
Agric Food Chem. 2007;55(15):6349-56.
442
[13] Oliveira J, Azevedo J, Silva AMS, Teixeira N, Cruz L, Mateus N, de Freitas V.
443
Pyranoanthocyanin dimers: a new family of turquoise blue anthocyanin-derived
444
pigments found in Port wine. J Agric Food Chem. 2010;58(8):5154-9.
445
[14] Oliveira J, Santos-Buelga C, Silva AMS, de Freitas V, Mateus N. Chromatic and
446
structural features of blue anthocyanin-derived pigments present in Port wine. Anal
447
Chim Acta. 2006;563(1-2):2-9.
448
[15] Oliveira J, Petrov V, Parola AJ, Pina F, Azevedo J, Teixeira N, Bras NF, Fernandes
449
PA, Mateus N, Ramos MJ, de Freitas V. Chemical behavior of methylpyranomalvidin-
450
3-O-glucoside in aqueous solution studied by NMR and UV-Visible spectroscopy. J
451
Phys Chem B. 2011;115(6):1538-45.
452
[16] Oliveira J, Mateus N, de Freitas V. Network of carboxypyranomalvidin-3-O-
453
glucoside (vitisin A) equilibrium forms in aqueous solution. Tetrahedron Lett.
454
2013;54(37):5106-10.
455
[17] Oliveira J, Mateus N, Silva AMS, de Freitas V. Equilibrium forms of Vitisin B
456
pigments in an aqueous system studied by NMR and Visible spectroscopy. J Phys Chem
457
B. 2009;113(32):11352-8.
458
[18] Schwarz M, Winterhalter P. A novel synthetic route to substituted
459
pyranoanthocyanins with unique colour properties. Tetrahedron Lett. 2003;44(41):7583-
460
7.
461
[19] Tron A, Gago S, McClenaghan ND, Parola AJ, Pina F. A blue 4',7-
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diaminoflavylium cation showing an extended pH range stability. Physical Chemistry
463
Chemical Physics. 2016;18(13):8920-5.
AC C
EP
TE D
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SC
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439
25
ACCEPTED MANUSCRIPT [20] Moncada MC, Fernandez D, Lima JC, Parola AJ, Lodeiro C, Folgosa F, Melo MJ,
465
Pina F. Multistate properties of 7-(N,N-diethylamino)-4'-hydroxyflavylium. An
466
example of an unidirectional reaction cycle driven by pH. Organic & Biomolecular
467
Chemistry. 2004;2(19):2802-8.
468
[21] Chassaing S, Isorez-Mahler G, Kueny-Stotz M, Brouillard R. Aged red wine
469
pigments as a source of inspiration for organic synthesis—the cases of the color-stable
470
pyranoflavylium
471
2015;71(20):3066-78.
472
[22] Oliveira J, Fernandes A, de Freitas V. Synthesis and structural characterization by
473
LC–MS and NMR of a new semi-natural blue amino-based pyranoanthocyanin
474
compound. Tetrahedron Lett. 2016;57(11):1277-81.
475
[23] Oliveira J, Araujo P, Fernandes A, Mateus N, de Freitas V. Synthesis and structural
476
characterization of amino-based pyranoanthocyanins with extended electronic
477
delocalization. Synlett. 2016;27:2459-62.
478
[24] Küster FW, Thiel A. Tabelle per le Analisi Chimiche e Chimico- Fisiche. 12 th ed.
479
Milano, Italy: Hoepli; 1982. p. 157-60.
480
[25] Trouillas P, Di Meo F, Gierschner J, Linares M, Sancho-García JC, Otyepka M.
481
Optical properties of wine pigments: theoretical guidelines with new methodological
482
perspectives. Tetrahedron. 2015;71(20):3079-88.
483
[26] Di Meo F, Sancho Garcia JC, Dangles O, Trouillas P. Highlights on Anthocyanin
484
Pigmentation and Copigmentation: A Matter of Flavonoid π-Stacking Complexation To
485
Be Described by DFT-D. Journal of Chemical Theory and Computation.
486
2012;8(6):2034-43.
flavylium-(4→8)-flavan
chromophores.
Tetrahedron.
AC C
EP
TE D
M AN U
SC
and
RI PT
464
26
ACCEPTED MANUSCRIPT [27] Anouar EH, Gierschner J, Duroux J-L, Trouillas P. UV/Visible spectra of natural
488
polyphenols: A time-dependent density functional theory study. Food Chem.
489
2012;131(1):79-89.
490
[28] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR,
491
Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X,
492
Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M,
493
Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H,
494
Vreven T, Montgomery Jr. JA, Peralta JE, Ogliaro F, Bearpark MJ, Heyd J, Brothers
495
EN, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell
496
AP, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam NJ, Klene M, Knox JE,
497
Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O,
498
Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski
499
VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas Ö,
500
Foresman JB, Ortiz JV, Cioslowski J, Fox DJ. Gaussian 09. Wallingford, CT, USA:
501
Gaussian, Inc.; 2009.
502
[29] Vallverdu-Queralt A, Biler M, Meudec E, Guerneve CL, Vernhet A, Mazauric JP,
503
Legras JL, Loonis M, Trouillas P, Cheynier V, Dangles O. p-Hydroxyphenyl-
504
pyranoanthocyanins: An Experimental and Theoretical Investigation of Their Acid-Base
505
Properties and Molecular Interactions. Int J Mol Sci. 2016;17(11).
506
[30] Carvalho ARF, Oliveira J, de Freitas V, Mateus N, Melo A. Unusual Color Change
507
of Vinylpyranoanthocyanin-Phenolic Pigments. J Agric Food Chem. 2010;58(7):4292-
508
7.
509
[31] Carvalho ARF, Oliveira J, de Freitas V, Mateus N, Melo A. A theoretical
510
interpretation of the color of two classes of pyranoanthocyanins. Theochem-J Mol
511
Struct. 2010;948(1-3):61-4.
AC C
EP
TE D
M AN U
SC
RI PT
487
27
ACCEPTED MANUSCRIPT 512
[32] Anouar EH, Osman CP, Weber J-FF, Ismail NH. UV/Visible spectra of a series of
513
natural and synthesised anthraquinones: experimental and quantum chemical
514
approaches. SpringerPlus. 2014;3:233.
515
[33] Oliveira J, Mateus N, de Freitas V. Previous and recent advances in
516
pyranoanthocyanins
517
2014;100(0):190-200.
in
aqueous
solution.
518
Dyes
and
Pigments.
RI PT
equilibria
ACKNOWLEDGEMENTS
520
This work received financial support from FEDER funds through COMPETE,
521
POPH/FSE, QREN and FCT (Fundação para a Ciência e Tecnologia) by a post-doctoral
522
scholarship (SFRH/BPD/112465/2015), two investigator contracts (IF/00225/2015 and
523
IF/01355/2014) and grants PTDC/AGR-TEC/2789/2014, REDE/1517/RMN/2005. This
524
work
525
POCI/01/0145/FEDER/007265) from FCT/MEC through national funds and co-
526
financed by FEDER, under the Partnership Agreement PT2020.
M AN U support
(UID/QUI/50006/2013
-
TE D
financial
EP
528
received
AC C
527
also
SC
519
28
ACCEPTED MANUSCRIPT 529
Supplementary material
530
1.200
1.000
RI PT
0.143 mM 0.114 mM
0.600
0.086 mM 0.057 mM
0.400
0.200
0.000 250
350
450
550
650
λ (nm)
SC
Absorbance
0.800
750
850
Figure 8 – UV-Visible spectra of pigment 3 in aqueous solution (15% (v/v) ethanol) at pH 6 at
533
different molar concentrations from 0.057 to 0.143 mM.
M AN U
531 532
AC C
EP
TE D
534
29
ACCEPTED MANUSCRIPT 20.000
0.057 mM
0.086 mM
0.114 mM
0.143 mM
18.000 16.000
Absorbance
14.000 12.000 10.000
6.000 4.000 2.000 0.000 250
350
450
550
650
λ (nm)
750
RI PT
8.000
850
Figure 9 – Normalized UV-Visible spectra of pigment 3 in aqueous solution (15% (v/v) ethanol)
537
at pH 6 at different molar concentrations from 0.057 to 0.143 mM.
M AN U
SC
535 536
AC C
EP
TE D
538
30
ACCEPTED MANUSCRIPT 2.5
1.5
1
0.5
0 350
450
550
650
750
λ (nm) 0s
2179 s
7185 s
850
SC
250
RI PT
Absorbance
2
Figure 10 – Spectral changes observed for pigment 3 upon pH jumps from a stock solution of pH
541
1 in 0.1M HCl to pH 6.42 with time (0, 2179 and 7185 s).
M AN U
539 540
AC C
EP
TE D
542 543
31
ACCEPTED MANUSCRIPT
SC
RI PT
544
M AN U
545 546
Figure 4 – Theoretical (TD-DFT) UV-visible absorption spectra of dicationic (A), cationic (B), neutral
547
(C) and anionic (D) forms of pigments 1, 2 and 3.
AC C
EP
TE D
548
32
SC
RI PT
ACCEPTED MANUSCRIPT
549
Figure 5 – MO correlation diagram of dicationic (A), cationic (B), neutral (C) and anionic (D) forms of
551
pigment 1.
M AN U
550
AC C
EP
TE D
552
33
SC
RI PT
ACCEPTED MANUSCRIPT
553
Figure 6 – MO correlation diagram of dicationic (A), cationic (B), neutral (C) and anionic (D) forms of
555
pigment 2.
M AN U
554
AC C
EP
TE D
556
34
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 7 – MO correlation diagram of dicationic (A), cationic (B), neutral (C) and anionic (D) forms of
559
pigment 3.
AC C
EP
TE D
M AN U
557 558
35
ACCEPTED MANUSCRIPT •
Equilibrium forms of dimethylamino-based pyranoanthocyanins in aqueous solutions. The absence of hydration reactions was confirmed by UV-Visible spectroscopy.
•
Structural features affected the protonation reaction at the amino group.
•
Structural features affected the deprotonation in the pyranoflavylium moiety.
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S0143-7208(17)30073-6
DOI:
10.1016/j.dyepig.2017.03.005
Reference:
DYPI 5831
To appear in:
Dyes and Pigments
Received Date: 12 January 2017 Revised Date:
1 March 2017
Accepted Date: 2 March 2017
Please cite this article as: Oliveira J, Araújo P, Fernandes A, Brás NF, Mateus N, Pina F, de Freitas V, Influence of the structural features of amino-based pyranoanthocyanins on their acid-base equilibria in aqueous solutions, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.03.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Influence of the structural features of amino-based pyranoanthocyanins on their
2
acid-base equilibria in aqueous solutions.
3 Joana Oliveira1,2*, Paula Araújo1, Ana Fernandes1, Natércia F. Brás3, Nuno Mateus1,
5
Fernando Pina4, Victor de Freitas1 1
6
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REQUIMTE – Laboratório Associado para a Química Verde, Departamento de
Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo
8
Alegre, 687, 4169-007 Porto, Portugal, 2
ICETA – Instituto de Ciências, Tecnologias e Agroambiente da Universidade do Porto,
10
Praça Gomes Teixeira, Apartado 55142, 4051-401 Porto, Portugal, 3
11 12
REQUIMTE – UCIBIO, Departamento de Química e Bioquímica, Faculdade de
Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal, 4
13
REQUIMTE – Laboratório Associado para a Química Verde, Departamento de
Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516
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Monte de Caparica, Portugal.
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*
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Tel: +351.220402596
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Author to whom correspondence should be addressed, [email protected]
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REQUIMTE
22
ICETA
23 24
ABSTRACT
1
ACCEPTED MANUSCRIPT The equilibrium forms of three different
families of dimethylamino-based
26
pyranoanthocyanins (1, 2 and 3) were studied in aqueous solutions at different pH
27
values from 1 to 12 using UV-Visible spectroscopy. The forms present under those
28
conditions are strongly correlated to the pyranoanthocyanin structural features. The
29
increase of the electronic delocalization helps the protonation at the amino group. At
30
very acidic pH condition (pH<0) the protonation at the amino group is observed for the
31
three pigments, but under less acidic conditions (pH~1) it only occurs for pigment 3
32
(pKa1=2.4±0.1) and at a lesser extent for pigment 2 (pKa1=1.1±0.1). At the same time,
33
the increase of the electronic delocalization on the amino-based pigments also favors the
34
deprotonation at the hydroxyl group present at carbon C-7 yielding the neutral quinoidal
35
base (pKa2=2.7±0.1, pKa2=4.8±0.1 and pKa2=5.4±0.1 for pigment 3, 2 and 1,
36
respectively). For pigment 3, the maximum molar fraction obtained for the
37
pyranoflavylium cation form is ~ 0.4 due to the proximity of the two acid-base
38
constants (pKa1 and pKa2) which indicates that at the pH range 1-5 three forms of the
39
compound are present in equilibrium (pyranoflavylium dication, pyranoflavylium cation
40
and neutral quinoidal base). The second deprotonation at the 4′-OH was less affected by
41
the structural features of the pigment with the ionization constant situated at pKa3~9
42
(pKa3=9.5±0.1, pKa3= 8.9±0.1 and pKa3=9.8±0.1 for pigment 1, 2 and 3, respectively).
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Keywords: amino-derived pyranoanthocyanins; vinylene linkage; butadienylidene linkage; protonation; deprotonation; UV-Visible; Density functional theory (DFT). 43 44
1. Introduction
45
Colour is the first quality parameter perceived in many products such as foodstuffs and
46
beverages, cosmetics, fabrics and others. Therefore, colorants (natural or synthetic) play
2
ACCEPTED MANUSCRIPT a crucial role to add value to the final product. Anthocyanins are natural pigments which
48
colour in aqueous solution is pH-dependent [1] and taking into account that the
49
hydration equilibrium constant (pKh) of anthocyanins is between 2 and 3, in the
50
majority of food matrices it is expected that anthocyanins occur largely as colorless
51
hemiketals (> 70%) in equilibrium with other forms.
52
Over the years, different families of anthocyanin-derived pigments have been described
53
in the literature, the majority being identified in wine matrices during the ageing
54
process, namely anthocyanin-flavanols (linked directly or mediated by aldehydes) [2-4],
55
A- and B-type vitisins [3], methylpyranoanthocyanins [5], oxovitisins [6],
56
acetylpyranoanthocyanins [7], pyranoanthocyanin-flavanols [8], pyranoanthocyanin-
57
phenols
58
Pyranoanthocyanins, the main anthocyanin-derived compounds found in nature, present
59
a vast palette of colours ranging from yellow to turquoise blue [6, 13-16], which can
60
constitute a challenging research field since natural blue colored pigments are rare in
61
nature. Moreover, studies in aqueous solutions using UV-Visible and NMR techniques
62
showed that pyranoanthocyanins present a higher colour stability when compared to
63
their anthocyanin precursors which can be explained by the absence of hydration
64
reactions in those anthocyanin-derivatives [15-17]. On the other hand, it has been
65
reported that the presence of a dimethylamino group in a cinnamyl moiety of a
66
pyranoanthocyanin compound created a bathochromic shift of ~40 nm when compared
67
to a similar compound containing two hydroxyl groups [18]. In fact, the presence of
68
amino groups in synthetic flavylium compounds (or flavylia) has already shown to
69
create an important bathochromic displacement in the maximum wavelength of the
70
compounds leading to the formation of bluish molecules [19-21].
10],
portisins
[11,
12]
and
pyranoanthocyanin
dimers
[13].
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3
ACCEPTED MANUSCRIPT 71
Bearing this, three different bluish amino-derived pyranomalvidin-3-O-glucoside
72
pigments were synthesized from the reaction of malvidin-3-O-glucoside with 4-
73
(dimethylamino)-cinnamic acid (Pigment 1) [18], carboxypyranomalvidin-3-O-
74
glucoside
75
methylpyranomalvidin-3-O-glucoside with 4-(dimethylamino)-cinnaldehyde (Pigment
76
3) [23] and their network of equilibrium forms were studied in aqueous solutions at
77
different pH from 1 to 12 by UV-Visible spectroscopy. The chromatic feature of these
78
amino-derived pyranoanthocyanins brings promising expectations concerning their use
79
in Industry as colorants and dyes.
4-(dimethylamino)-cinnamic
acid
(Pigment
2)
[22]
and
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2. Material and Methods
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2.1. Reagents
83
pH 1.00 (chloride acid/potassium chloride), 4.00 (sodium citrate) and 7.00 (sodium
84
phosphate) buffer solutions were obtained from Fluka (Madrid, Spain). A universal
85
buffer (1 L) of Theorell and Stenhagen [24] was prepared dissolving 2.25 mL of
86
phosphoric acid 85% (w/w), 7.00 g of monohydrated citric acid, 3.54 g of boric acid and
87
343 mL of a 1 M NaOH solution.
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2.2. Hemi-synthesis of amino based malvidin-3-O-glucoside pigments 1, 2 and 3
90
4-(Dimethylamino)-cinnamyl-pyranomalvidin-3-O-glucoside- (1) was obtained from
91
the reaction of malvidin-3-O-glucoside with 4-(Dimethylamino)-cinnamic acid
92
according to the procedure reported in the literature [18]. 4-(Dimethylamino)-cinnamyl-
93
10-vinylene-pyranomalvidin-3-O-glucoside (2) was synthesized from the reaction of the
94
carboxypyranomalvidin-3-O-glucoside with 4-(Dimethylamino)-cinnamic acid as
95
described
previously
[22].
4-(Dimethylamino)-cinnamyl-10-butadienylidene-
4
ACCEPTED MANUSCRIPT 96
pyranomalvidin-3-O-glucoside (3) pigment was obtained from the reaction of the
97
methylpyranomalvidin-3-O-glucoside with
98
reported elsewhere [23]. The purity of all pigments was confirmed by NMR.
4-(Dimethylamino)-cinnamaldehyde as
99 2.3. Titration of pigments 1, 2 and 3 by UV-Visible spectroscopy
101
Stock solutions (0.24 mM) were prepared for pigments 1, 2 and 3 in an aqueous solution
102
of 45% (v/v) ethanol (to obtain a final concentration of 15% (v/v) ethanol) 0.1 M HCl. 1
103
mL of a 0.1 M NaOH solution was added to a 10x10 mm quartz cell, 1 mL of Theorell
104
and Stenhagen universal buffer solution at pH~1 and 1 mL of the stock solution (the
105
pigment final concentration was 0.08 mM). The titrations of the pigments were
106
performed until pH~12 by the addition of small volumes (1-5 µL) of base (1 M or 10 M
107
NaOH). For each pigment, the first spectrum (250-900 nm) was run immediately after
108
the addition of the stock solution and shaking the cell. Successive spectra were recorded
109
instantly after the addition of the base in a Thermo Scientific Evolution Array UV-
110
Visible spectrophotometer at 25ºC. The obtained spectra were adjusted for the final
111
volume in each point. The pH values of the initial solution and after the addition of the
112
base were measured in a WTW pH 320 (Weilheim, Germany) with a CRISON 5209
113
combined glass electrode of 3 mm diameter (Barcelona, Spain). The pH meter was
114
calibrated with pH 1 and 4 buffer solutions for pH values below 2.5 and, pH 4 and 7
115
buffer solutions for pH values above 2.5. The fittings for pKa’s determination were
116
carried out using the Solver program from Microsoft Excel.
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117 118
2.4. Theoretical calculation for UV-visible properties
119
Density functional theory (DFT) calculations with the hybrid functional B3P86 and the
120
basis set 6-31+G(d,p) were used to optimize the geometry of the twelve
5
ACCEPTED MANUSCRIPT pyranoanthocyanin equilibrium forms (dicationic, cationic, neutral quinoidal and
122
anionic quinoidal forms for each pigment). This functional and basis set were
123
specifically chosen due to its good ability to calculate thermodynamic and UV-visible
124
absorption properties for polyphenols [25-27]. Frequency calculations were carried out
125
at the same level of theory in order to confirm the absence of imaginary frequencies in
126
the ground state. Subsequently, Time Dependent (TD) DFT single-point calculations
127
with the same level of theory were applied to determine the excited singlet state
128
energies, and subsequently, the allowed vertical π → π∗ electronic excitation energies
129
were obtained. These provide the absorption energies in the UV-visible range with the
130
contribution of all one-electron transitions and their oscillator strength.
131
The TD-DFT calculations were performed in vacuum and in solvent. For continuum
132
solvent calculations, the integral equation formalism Polarizable Continuum Model
133
(IEFPCM) method with a dielectric constant (ε) of 80 was used. This value of ε allows
134
to evaluate the long-range effect of an aqueous environment. All calculations were
135
carried out with the Gaussian09 software package [28].
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3. Results and discussion
138
The equilibrium forms of three families of amino-based pyranoanthocyanins (Figure 1)
139
were evaluated in aqueous solutions at different pH values from 1 to 12 by UV-Visible
140
spectroscopy.
141
pyranomalvidin-3-O-glucoside moiety linked to a 4-(dimethylamino)-cinnamyl group.
142
The structural differences between the three pigments are in the type of linkage
143
preforming the connection between both moieties. In pigment 1 the moieties are linked
144
directly through a C – C bond. In the case of pigment 2 the connection is made through
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The
studied
compounds
are
similar
molecules
containing
a
6
ACCEPTED MANUSCRIPT a vinylene linkage and in pigment 3 through a butadienylidene one. These structural
146
features confer to the pigments different colours and stabilities in aqueous solutions.
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Figure 1 – Structure of the three amino-derived pyranoanthocyanin pigments in their
150
pyranoflavylium cation form.
151
3.1. Equilibrium forms in aqueous solutions at different pH values
153
3.1.1. 4-(dimethylamino)-cinnamyl-10-pyranomalvidin-3-O-glucoside (1)
154
The maximum absorption wavelength for pigment 1, in aqueous solution at acidic pH
155
value (pH 1.99) is 557 nm and the compound displays a violet colour. With the increase
156
of the pH value until 6.13 a decrease in the absorbance at the maximum wavelength is
157
observed together with a hypsochromic shift and the appearance of a shoulder around
158
500 nm that increases with pH (Figure 2A). This was attributed to the equilibrium
159
between the pyranoflavylium cation form and the neutral quinoidal base. This
160
equilibrium is confirmed by the presence of an inflection point in the titration curve of
161
the absorbance at 557 nm versus the pH in this pH range (Figure 3A). Although it is not
162
usual to observe quinoidal bases of pyranoanthocyanins that absorb close to 500 nm,
163
this behavior has already been reported in the literature by Schwarz and Winterhalter for
164
this compound [18], by Oliveira et al. for similar compounds [12, 14] and more recently
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ACCEPTED MANUSCRIPT by Vallverdú-Queralt et al. for two pyranoanthocyanins [29]. For higher pH values up
166
to ~12, it is observed the increase of the absorbance at 500 nm and the appearance of a
167
shoulder at 600 nm (Figure 2B). This trend can be due to the equilibrium between the
168
pyranoflavylium neutral quinoidal form and the respective anionic form that is
169
supported by the second inflection point observed in the titration curve in this pH range
170
(Figure 3A).
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pH 1.99 1.6
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pH 6.13
1 0.8 0.6 0.4
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1.2
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pH 11.85
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Absorbance
0.8
pH 7.59
0.6
AC C
0.4
B
0.2
0
250
171
350
450
550
650
750
850
λ (nm)
172
Figure 2 – UV-Visible spectra of pigment 1 in aqueous solution at different pH values A: from
173
1.99 to 6.13; B: from 7.59 to 11.85.
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ACCEPTED MANUSCRIPT 1.8 1.6
A
Absorbance
1.4 1.2
pKa2 = 5.4±0.1
1.0 0.8
557 nm 470 nm
pKa3 = 9.5±0.1
0.6
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7 pH
B
pKa1 = 1.1±0.1
pKa2 = 4.8±0.1
420 nm
3.00
5.00
7.00 pH
11.00
13.00
C
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9.00
510 nm
pKa3 = 9.8±0.1
pKa2 = 2.7±0.1
1 0.5
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665 nm
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Absorbance
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520 nm
pKa3 = 8.9±0.1
2
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11
551 nm
2.5
1.5
9
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1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1.00
3
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3.00
5.00
7.00 pH
9.00
11.00
13.00
175
Figure 3 – Titration curves of the absorbance at different wavelengths versus pH and respective
176
pKa ’s at T=298 K at pH 1–12 for A: for pigment 1; B: for pigment 2; C: for pigment 3.
177 178
3.1.2. 4-(dimethylamino)-cinnamyl-10-vinylene-pyranomalvidin-3-O-glucoside (2)
179
In the case of pigment 2, the UV-Visible spectrum in acid conditions (pH 1.44) showed
180
a maximum absorption wavelength at 520 nm (Figure 4A). Under these conditions this 9
ACCEPTED MANUSCRIPT compound presents a red colour. With the increase of the pH up to 2.58 a decrease in
182
the absorbance at this wavelength is observed concomitantly with an increase at 633 nm
183
(Figure 4A). The first assumption that was made was that this would correspond to the
184
equilibrium between the pyranoflavylium cation form and the respective neutral
185
quinoidal form. However, the spectrum of the same compound determined at very low
186
pH values (pH=0, -0.5 and -0.7) showed the disappearance of the shoulder at 633 nm
187
and at the same time the increase of the absorbance at 520 nm (Figure 4A). The spectra
188
obtained for the solutions in very acidic conditions is similar to the one reported for the
189
pigment at pH 1.44. This indicates that the equilibrium forms that should be present at
190
these pH values are the protonated form (at the amino group) of the compound
191
(dication) in equilibrium with the respective pyranoflavylium cation form. In fact, the
192
protonation of amino groups has already been reported in the literature for synthetic
193
flavyliums [19, 20]. The detection of the pyranoflavylium dication form of pigment 2 by
194
ESI-MS was not possible to observe due to its low pKa1 value making difficult its
195
detection in the mass spectrum under the conditions used, contrarily to pigment 3 as
196
discussed below. The protonation of the amino group leads to an interruption in the
197
electronic delocalization of the molecule, explaining why although pigment 2 has higher
198
electronic delocalization than pigment 1 it presents an unexpected red colour in acidic
199
conditions.
200
With the increase of the pH of the solution for values up to 8.20 a reverse trend is
201
observed, with a decrease in the absorbance at 633 nm at the same time as the increase
202
of the absorbance at ~520 nm (Figure 4B). This corresponds to the equilibrium between
203
the pyranoflavylium cation form and the respective neutral quinoidal form. The titration
204
curve of the absorbance at 420, 520 and 551 nm presented in Figure 3A shows an
205
inflection point corresponding to this equilibrium. Moreover, at the pH range 7-12 is
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10
ACCEPTED MANUSCRIPT 206
observed the increase of the absorbance at ~520 nm and the equilibrium between the
207
neutral and the anionic quinoidal forms is observed in a similar manner as described for
208
pigment 1. In this pH range it is also detected an inflection point in the titration curves
209
(Figure 3A) confirming the postulated equilibrium.
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0.6
0.5 pH - 0.7 pH - 0.5
pH 2.58
pH 0
0.3
0.2
A
pH 1.44
0.1
0 350
450
550 λ (nm)
1.6
650
pH 2.71
1.4
1.2
pH 8.20
0.8
850
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Absorbance
1
750
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0.6
0.4
0 250
350
EP
0.2
450
550
650
SC
Aborbance
0.4
750
B 850
λ (nm)
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1.6
pH 11.77
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Absorbance
1.2
1
pH 7.16
0.8
0.6
0.4
C
0.2
0 250
210
350
450
550
650
750
850
λ (nm)
11
ACCEPTED MANUSCRIPT 211
Figure 4 – UV-Visible spectra of pigment 2 in aqueous solution at different pH values A: from -
212
0.7 to 2.44; B: from 2.71 to 8.20; C: at 7.16 to 11.77.
213 3.1.3. 4-(dimethylamino)-cinnamyl-10-butadienylidene-pyranomalvidin-3-O-glucoside
215
(3)
216
For pigment 3, the UV-Visible spectra recorded under acidic conditions (pH 1.60)
217
showed a maximum absorption wavelength at 535 nm (Figure 5A). Since this pigment
218
presents higher electronic delocalization than pigments 1 and 2 it was expected to
219
present a more bluish colour. However, in acid conditions the pigment presents a red
220
colour and is hence thought to be present mainly in its dication pyranoflavylium form,
221
similarly to pigment 2 but in a greater extent. Moreover, the spectrum at pH 1.60 is very
222
similar to the one observed at very acidic conditions (pH 0, -0.5 and -0.7) (Figure 5A).
223
In fact, the presence of the dicationic form of this compound was already confirmed by
224
ESI-MS with m/z=m/2=344.67 in the positive ion mode [23]. With the increase of the
225
pH value up to 2.65 it is observed the decrease in the absorbance at this maximum
226
absorption wavelength together with the increase at 665 nm (Figure 5A). This was
227
attributed to the equilibrium between the pyranoflavylium dication and the cation forms.
228
For pH values between 2.67 and 5.68 a third absorption wavelength at 588 nm appears
229
and decreases in intensity with the increase of the pH (Figure 5B). This indicates that at
230
this pH range additionally to the pyranoflavylium dication and cation forms it is also
231
observed the presence of neutral quinoidal form. This is confirmed by the two inflexion
232
points observed in the titration curve of the absorbance at 510 and 665 nm versus the
233
pH (Figure 5B) at this pH range.
234
Contrarily to catechyl-pyranomalvidin-3-O-glucoside and guaiacyl-pyranomalvidin-3-
235
O-glucoside pigments that were reported by Vallverdú-Queralt et al. to be prone to
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12
ACCEPTED MANUSCRIPT aggregation above pH 4 when the neutral quinoidal base is mainly present [29], pigment
237
3 does not seem to aggregate at the concentration used in this study. In fact, the shape of
238
the spectra obtained for the pigment at pH 6.5 (where the neutral quinoidal base is
239
present) at different molar concentrations from 0.057 to 0.143 mM (Figure 1 –
240
Supplementary material) is similar. Moreover, by normalizing the spectra obtained by
241
the division of the absorbance at each wavelength by the molar concentration it is
242
possible to observe an almost complete juxtaposition of the spectra which also
243
corroborates with the absence of aggregation (Figure 2 – Supplementary material). In
244
addition, in Figure 3 (Supplementary material) it is also possible to observe only a
245
small change in the absorption spectra of pigment 3 with time (0, 2179 and 7185 s) at
246
pH 6.42.
247
For higher pH values up to 11.53, the absorbance at 535 nm increases along with the
248
decrease at 588 and 665 nm (Figure 5C). This tendency is due to the equilibrium
249
between the neutral and the anionic quinoidal forms.
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ACCEPTED MANUSCRIPT 1 0.9 0.8 pH -0.7 pH -0.5 pH 0
0.6 0.5 0.4
A
pH 2.65
0.3 0.2
pH 1.60
0.1
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Absorbance
0.7
0 250
350
450
550
650
750
850
λ (nm) 2
pH 2.41
SC
pH 2.67
pH 5.87 1
B
0.5
0 250
350
450
550
650
λ (nm) pH 11.53 2
850
pH 5.87
1
0 250
350
EP
0.5
450
550
650
750
C 850
λ (nm)
AC C
250
750
TE D
Absorbance
1.5
M AN U
Absorbance
1.5
251
Figure 5 – UV-Visible spectra of pigment 3 in aqueous solution at different pH values A: from -
252
0.7 to 2.65; B: from 2.41 to 5.87; C: from 5.87 to 11.53.
253 254
3.2. TD-DFT calculations
255
The UV-visible absorption spectra and the Molecular Orbitals (MO) correlation
256
diagrams for the four forms [dicationic (A), cationic (B), neutral (C) and anionic (D)] of
257
pigments 1, 2 and 3 were also determined by TD-DFT calculations (Figures 4 to 7 in
14
ACCEPTED MANUSCRIPT Supplementary material). The dicationic form of pigment 1 was predicted using TD-
259
DFT calculation as corresponding to the first excited state S0 → S1 (at 504.73 nm),
260
which is essentially related to the HOMO → LUMO electronic transition. Moreover,
261
the experimental large band at 557 nm (Figure 2A) exhibited by the cationic form of
262
pigment 1 which is predicted theoretically as the second excited state (S0 → S2) at
263
446.74 nm (Figure 5 in Supplementary material). This corresponds to the HOMO-1 →
264
LUMO electronic transition, in which both MO are being delocalized along the
265
extended conjugated path of the dimethylamino-cinnamyl-ring. Although the LUMO is
266
almost similar for dicationic and cationic forms, the first deprotonation induces a
267
stabilization of the HOMO and HOMO-1 due to the large delocalization occurred in the
268
dimethylamino-cinnamyl -ring, and subsequently decreases the energy gap and results
269
in a slight bathochromic shift. Concerning the neutral and anionic forms, the
270
experimental large band at 500 nm and shoulder at 600 nm are theoretically predicted
271
by the first excited states at 468.77 nm and 569.49 nm, respectively, which still ascribe
272
to the HOMO → LUMO electronic transition. The HOMO of neutral form is mainly
273
located at A-ring and dimethylamino-cinnamyl -ring, while in the anionic form it was
274
displaced from the dimethylamino-cinnamyl-ring to the B-ring.
275
The bands of dicationic and cationic forms of pigment 2 predicted by the TD-DFT
276
calculations correspond to the first excited state S0 → S1 (at 468.48 nm and 465.54 nm,
277
respectively) that is defined by the HOMO → LUMO electronic transition. As observed
278
in pigment 1, the LUMO is almost identical for both forms, whereas the HOMO is
279
severely displaced toward the amino-ring, which dramatically decreases the overlap
280
with the LUMO. This strong concentration of HOMO around the dimethylamino-
281
cinnamyl-ring (see Figure 6 of Supplementary material) occurs due to the higher
282
delocalization expected on this compound, in comparison with pigment 1. In relation to
AC C
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SC
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258
15
ACCEPTED MANUSCRIPT the neutral form, the TD-DFT predicts the first excited state at 481.03 nm that is
284
attributed to the HOMO → LUMO electronic transition, and both MO are mainly
285
concentrated in the A-ring. For the anionic form, the first excited state (corresponding to
286
the HOMO → LUMO electronic transition) was predicted at 540.36 nm. The wider
287
distribution of HOMO-1 than HOMO around the A-ring and B-ring is related to the
288
higher delocalization verified in this form, which justifies the higher wavelength and the
289
expectation of its bluish colour.
290
The theoretical UV-visible absorption spectra predicted for pigment 3 are very similar
291
to the ones observed for pigment 2. The dicationic, cationic, neutral and anionic have
292
first excited states (HOMO → LUMO electronic transition) at 465.44 nm, 464.54 nm,
293
480.40 nm and 538.36 nm, respectively. However, the cationic form of pigment 3 has
294
both HOMO and HOMO-1 mostly located along the amino group (Figure 7 of
295
Supplementary material), which indicates that the second excited state associated to the
296
HOMO-1 → LUMO electronic transition is also involved in the bluer colour of cationic
297
form than dicationic form. For anionic form, the HOMO → LUMO electronic transition
298
predicted at 538.36 nm (corresponding to the experimental band at 535 nm) is related to
299
the delocalization verified around the A-ring and B-ring.
300
Theoretical and experimental spectra do not overlap completely, especially those
301
concerning the dicationic pyranoflavylium forms and the anionic quinoidal forms.
302
However, it is not the first time that unusual chromatic features have been reported in
303
the literature for other pyranoanthocyanins, like the colour change from violet to blue
304
with the decrease of the temperature from 25ºC to -10ºC for aqueous solutions of
305
pyranoanthocyanin-vinyl-cinnamyl (Portisin B) pigments.
306
chemical change was explained to be due to electronic and vibrational effects [30].
307
Moreover, the hypsochromic shift observed in the maximum absorption wavelength for
AC C
EP
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SC
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283
This reversible physical-
16
ACCEPTED MANUSCRIPT pyranoanthocyanins in general (e.g. A-type and B-type vitisins) when compared to the
309
anthocyanin counterpart although they present a higher electronic delocalization was
310
explained using quantum theoretical studies by differences in the planarity of the
311
ubiquitous B ring in this kind of pigments [31]. The optimized geometry of quinoidal
312
anionic forms have a lesser planarity of the B-ring than the other forms (data not shown)
313
and that could explain the hypsochromic shift observed in the maximum absorption
314
wavelength for these equilibrium forms in aqueous solutions. In addition, the dicationic
315
pyranoflavylium forms also revealed a distortion from the planarity of the
316
dimethylaminocinnamyl-ring that should be due to the presence of the proton at the
317
dimethylamino group. Here, we only determined the UV-Vis spectrum for one
318
optimized conformation for each form of the three pigments. More precise
319
measurements (with a structural conformational study to determine the most
320
energetically stable conformation of each compound, and then calculate their UV-Vis
321
spectra or the average wavelength values of all conformations) could be required to
322
obtain more reliable data. However, they would represent a highly demanding approach
323
(in terms of computation time) considering the high number of forms and compounds,
324
as well as the large number of rotatable bond present in each molecule. On the other
325
hand, the DFT has an error associated, in particular the density functional B3P86 has a
326
deviation in the determination of maximum wavelength values of natural molecules
327
between 11 nm [25] and 24 nm [32]. Although this error is smaller than the one
328
obtained with other density functionals, it may justify, for example the discrepancy
329
occurred in the wavelength maximum values of dicationic and cationic forms of
330
pigments 2 and 3 (exp: cation > dication and teor: cation ≈ dication). Overall, a
331
combination of these effects may be responsible for the differences observed for some
332
theoretical and experimental spectra.
AC C
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308
17
ACCEPTED MANUSCRIPT 333 334
3.3. Structural features versus acid-base equilibria
335
Based
336
pyranoanthocyanins at different pH values from 1 to 12 it was possible to determine the
337
type of equilibria that were taking place in aqueous solutions for each pigment. In
338
general, pyranoanthocyanins can undergo two deprotonations at 7-OH from ring A and
339
at 4′-OH from ring B [15, 33]. In the case of pigments containing amino groups, an
340
additional protonation at this moiety can occur at acidic conditions [19, 20]. However,
341
in this study, it was demonstrated that the presence of an amino group and its distance to
342
the pyranoanthocyanin moiety has a great influence on the acid-base equilibria in
343
aqueous solutions of the pigments. According to the results discussed previously, in
344
aqueous solutions from pH 1 to 12, pigment 1 is present in three equilibrium forms, the
345
pyranoflavylium, the neutral and anionic quinoidal forms. In the case of pigments 2 and
346
3, the pyranoflavylium dication form is also present additionally to the other three
347
(Figure 6). After defining the particular equilibria forms for each pigment, the acid-base
348
ionization constants were determined, plotting the absorbance at selected wavelengths
349
as a function of the pH of the solution (Figure 4). The fittings for determination of the
350
pKa values were carried out using the Solver program from Microsoft Excel and are
351
presented in Table 1.
the
UV-Visible
spectra
obtained
for
the
three
amino-derived
AC C
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on
18
ACCEPTED MANUSCRIPT OCH3
OCH3
A
OH
OH
HO
HO
O
O OCH3
OCH3 O HO
OH OH
O
OH
O HO
OH
O
OH
O
OH
O
pKa1
H
H3C
CH3
CH3
RI PT
N
N H3C
H+
+ n = 0, 1, 2
n = 0, 1, 2
OCH3
OCH3
OH
OH O HO
SC
B
O
OCH3
O OCH3 OH
O
OH
O
O
O
M AN U
O HO
OH
O HO
OH
OH OH
+
H+
+
H+
pKa2
n = 0, 1, 2
n = 0, 1, 2
N
H3C
N CH 3 OCH3
C
TE D
H3C
CH3
OCH3
OH O
O
O O
O
OCH3 O HO
O
O HO
OH
OH
EP
O
OCH3
OH
pKa3
AC C
n = 0, 1, 2
N
H3C
CH3
O
OH OH
O
n = 0, 1, 2
352 353
OH
N H3C
CH3
354
Figure 6 – General representation of the proton transfer equilibria of the amino-derived
355
pyranoanthocyanins 1 (n=0), 2 (n=1) and 3 (n=2) in aqueous solutions at pH between 1 and 12. )
356 357
19
ACCEPTED MANUSCRIPT 358
Table 1 – pK a values of the three amino-derived pyranoanthocyanins determined by UV-Visible.
359
E, λ max determined in ethanol 0.01% HCl; λ max of AH 2 2+, AH + , A and A- determined from the
360
experimental data (s, indicates a shoulder). λmax (nm)
pKa
E
AH22+
AH+
A
A-
1
562
n.d.
557
512
509
632
519
633
623, 519
543, 612
(s)
(s)
554
531, 612
2
668
531
625
<1
pKa2
pKa3
5.4±0.1
9.5±0.1
1.1±0.1 4.8±0.1
8.9±0.1
2.4±0.1 2.7±0.1
9.8±0.1
SC
3
pKa1
RI PT
Pigment
(s)
M AN U
361
The acid-base constants presented in Table 1 indicate that the protonation at the amino-
363
group and the deprotonation at the hydroxyl group present in carbon C-7 from ring A
364
are the most affected equilibria by the pigments structural features. Pyranoanthocyanins
365
linked directly to the 4-(dimethylamino)-cinnamyl (pigment 1) are more resistant to the
366
protonation (pKa1<1) at the amino group than pyranoanthocyanins linked to the 4-
367
(dimethylamino)-cinnamyl by a vinylene group (pigment 2) (pKa1=1.1±0.1) or by a
368
butadienylidene one (pigment 3) (pKa1=2.4±0.1). This latter was the most susceptible to
369
the protonation reaction presenting a lower ionization constant. In a similar manner, the
370
deprotonation reaction at 7-OH from the ring A of the pyranoflavylium cation to yield
371
the neutral quinoidal form is correlated to the pigment electronic delocalization. It
372
occurs more easily for pigment 3 (pKa2=2.7±0.1) with higher electronic delocalization,
373
than for pigment 2 (pKa1=4.8±0.1) and pigment 1 (pKa2=5.4±0.1). On the other hand,
374
the second deprotonation at 4′-OH is the less affected acid-base equilibrium by the
375
structural features of the molecule (pigments 1, 2 and 3 with pKa3=9.5±0.1,
376
pKa3=8.9±0.1 and pKa3=9.8±0.1). This tendency can be easily observed in the molar
AC C
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362
20
ACCEPTED MANUSCRIPT fraction of the network of equilibrium forms for the three pigments studied presented in
378
Figure 7.
379
It seems that the presence of a butadienylidene linkage (pigment 3) in a
380
pyranoanthocyanin compound stabilizes the deprotonated forms of the compound when
381
compared with similar compounds presenting a vinylene group (pigment 2). This has
382
already been observed in a minor extent for similar pigments containing a sinapyl
383
moiety linked to pyranoanthocyanins [33].
AC C
EP
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M AN U
SC
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377
21
ACCEPTED MANUSCRIPT 1.0
AH+
0.9
A-
A
Molar fraction
0.8 0.7 0.6 0.5 0.4 0.3
A
0.1 0.0 1
3
5
7
9
11
pH 1.0 0.9
A-
A
AH+
SC
0.7 0.6 0.5 0.4 0.3
AH22+
0.2
B
0.1 0.0 1.00
3.00
5.00
7.00
pH 1.0
AH22+
9.00
0.6
AH+
11.00
A-
EP
0.4
TE D
A
0.8
Molar fraction
M AN U
Molar fraction
0.8
RI PT
0.2
0.2
C
AC C
0.0
1.00
3.00
5.00
7.00
9.00
11.00
pH
384 385
Figure 7 – Molar fraction distribution diagram for pigment A: 1; B: 2 and C: 3 as a function of the
386
pH obtained from the UV-Visible determination.
387 388
4. Conclusions
389
The structural differences concerning the three pyranomalvidin-3-O-glucoside
390
derivatives is in the type of linkage performing the connection between the pyran 22
ACCEPTED MANUSCRIPT moiety and the dimethylaminophenyl unit yielding pigments with different structural
392
features and electronic delocalization. It was observed by UV-Visible spectroscopy that
393
the forms present in aqueous solutions at different pH values from 1 to 12 are strongly
394
correlated to the pyranoanthocyanin structural features. It seems that the increase of the
395
electronic delocalization helps the protonation at the amino group since it was observed
396
that the protonation was favored for pigment 3, the one that presents a higher electronic
397
delocalization and then for pigment 2. This kind of equilibrium was not observed for
398
pigment 1 in the conditions studied.
399
With respect to the color stability and concerning the possible applications of these
400
pigments as colorants, it appears that pigment 3 is the less viable since at the pH of most
401
food matrices (pH 2-5) this pigment is present in three different forms in equilibrium
402
with the pyranoflavylium cation form (blue color) accounting for only 40% (Figure
403
7C). The most stable compound at those pH values is pigment 1 (Figure 7A) although
404
the color presented is not the most interesting one.
405
TE D
M AN U
SC
RI PT
391
5. Literature cited
407
[1] Brouillard R, Dubois J-E. Mechanism of the structural transformations of
408
anthocyanins in acidic media. J Am Chem Soc. 1977;99(5):1359-64.
409
[2] Pissarra J, Mateus N, Rivas-Gonzalo J, Buelga CS, de Freitas V. Reaction between
410
malvidin 3-glucoside and (+)-catechin in model solutions containing different
411
aldehydes. J Food Sci. 2003;68(2):476-81.
412
[3] Salas E, Atanasova V, Poncet-Legrand C, Meudec E, Mazauric JP, Cheynier V.
413
Demonstration of the occurrence of flavanol-anthocyanin adducts in wine and in model
414
solutions. Anal Chim Acta. 2004;513(1):325-32.
AC C
EP
406
23
ACCEPTED MANUSCRIPT [4] Salas E, Le Guerneve C, Fulcrand H, Poncet-Legrand C, Cheynier W. Structure
416
determination and colour properties of a new directly linked flavanol-anthocyanin
417
dimer. Tetrahedron Lett. 2004;45(47):8725-9.
418
[5] He J, Santos-Buelga C, Silva AMS, Mateus N, De Freitas V. Isolation and structural
419
characterization of new anthocyanin-derived yellow pigments in aged red wines. J Agric
420
Food Chem. 2006;54(25):9598-603.
421
[6] He J, Oliveira J, Silva AMS, Mateus N, De Freitas V. Oxovitisins: a new class of
422
neutral pyranone-anthocyanin derivatives in red wines. J Agric Food Chem.
423
2010;58(15):8814-9.
424
[7] Gomez-Alonso S, Blanco-Vega D, Victoria Gomez M, Hermosin-Gutierrez I.
425
Synthesis, isolation, structure elucidation, and color properties of 10-acetyl-
426
pyranoanthocyanins. J Agric Food Chem. 2012;60(49):12210-23.
427
[8] He J, Santos-Buelga C, Mateus N, de Freitas V. Isolation and quantification of
428
oligomeric pyranoanthocyanin-flavanol pigments from red wines by combination of
429
column chromatographic techniques. Journal of Chromatography A. 2006;1134(1-
430
2):215-25.
431
[9] Schwarz M, Jerz G, Winterhalter P. Isolation and structure of pinotin A, a new
432
anthocyanin derivative from Pinotage wine. Vitis. 2003;42(2):105-6.
433
[10] Schwarz M, Wabnitz TC, Winterhalter P. Pathway leading to the formation of
434
anthocyanin−vinylphenol adducts and related pigments in red wines. J Agric Food
435
Chem. 2003;51(12):3682-7.
436
[11] Mateus N, Silva AMS, Rivas-Gonzalo JC, Santos-Buelga C, De Freitas V. A new
437
class of blue anthocyanin-derived pigments isolated from red wines. J Agric Food
438
Chem. 2003;51(7):1919-23.
AC C
EP
TE D
M AN U
SC
RI PT
415
24
ACCEPTED MANUSCRIPT [12] Oliveira J, de Freitas V, Silva AMS, Mateus N. Reaction between
440
hydroxycinnamic acids and anthocyanin-pyruvic acid adducts yielding new portisins. J
441
Agric Food Chem. 2007;55(15):6349-56.
442
[13] Oliveira J, Azevedo J, Silva AMS, Teixeira N, Cruz L, Mateus N, de Freitas V.
443
Pyranoanthocyanin dimers: a new family of turquoise blue anthocyanin-derived
444
pigments found in Port wine. J Agric Food Chem. 2010;58(8):5154-9.
445
[14] Oliveira J, Santos-Buelga C, Silva AMS, de Freitas V, Mateus N. Chromatic and
446
structural features of blue anthocyanin-derived pigments present in Port wine. Anal
447
Chim Acta. 2006;563(1-2):2-9.
448
[15] Oliveira J, Petrov V, Parola AJ, Pina F, Azevedo J, Teixeira N, Bras NF, Fernandes
449
PA, Mateus N, Ramos MJ, de Freitas V. Chemical behavior of methylpyranomalvidin-
450
3-O-glucoside in aqueous solution studied by NMR and UV-Visible spectroscopy. J
451
Phys Chem B. 2011;115(6):1538-45.
452
[16] Oliveira J, Mateus N, de Freitas V. Network of carboxypyranomalvidin-3-O-
453
glucoside (vitisin A) equilibrium forms in aqueous solution. Tetrahedron Lett.
454
2013;54(37):5106-10.
455
[17] Oliveira J, Mateus N, Silva AMS, de Freitas V. Equilibrium forms of Vitisin B
456
pigments in an aqueous system studied by NMR and Visible spectroscopy. J Phys Chem
457
B. 2009;113(32):11352-8.
458
[18] Schwarz M, Winterhalter P. A novel synthetic route to substituted
459
pyranoanthocyanins with unique colour properties. Tetrahedron Lett. 2003;44(41):7583-
460
7.
461
[19] Tron A, Gago S, McClenaghan ND, Parola AJ, Pina F. A blue 4',7-
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diaminoflavylium cation showing an extended pH range stability. Physical Chemistry
463
Chemical Physics. 2016;18(13):8920-5.
AC C
EP
TE D
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SC
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439
25
ACCEPTED MANUSCRIPT [20] Moncada MC, Fernandez D, Lima JC, Parola AJ, Lodeiro C, Folgosa F, Melo MJ,
465
Pina F. Multistate properties of 7-(N,N-diethylamino)-4'-hydroxyflavylium. An
466
example of an unidirectional reaction cycle driven by pH. Organic & Biomolecular
467
Chemistry. 2004;2(19):2802-8.
468
[21] Chassaing S, Isorez-Mahler G, Kueny-Stotz M, Brouillard R. Aged red wine
469
pigments as a source of inspiration for organic synthesis—the cases of the color-stable
470
pyranoflavylium
471
2015;71(20):3066-78.
472
[22] Oliveira J, Fernandes A, de Freitas V. Synthesis and structural characterization by
473
LC–MS and NMR of a new semi-natural blue amino-based pyranoanthocyanin
474
compound. Tetrahedron Lett. 2016;57(11):1277-81.
475
[23] Oliveira J, Araujo P, Fernandes A, Mateus N, de Freitas V. Synthesis and structural
476
characterization of amino-based pyranoanthocyanins with extended electronic
477
delocalization. Synlett. 2016;27:2459-62.
478
[24] Küster FW, Thiel A. Tabelle per le Analisi Chimiche e Chimico- Fisiche. 12 th ed.
479
Milano, Italy: Hoepli; 1982. p. 157-60.
480
[25] Trouillas P, Di Meo F, Gierschner J, Linares M, Sancho-García JC, Otyepka M.
481
Optical properties of wine pigments: theoretical guidelines with new methodological
482
perspectives. Tetrahedron. 2015;71(20):3079-88.
483
[26] Di Meo F, Sancho Garcia JC, Dangles O, Trouillas P. Highlights on Anthocyanin
484
Pigmentation and Copigmentation: A Matter of Flavonoid π-Stacking Complexation To
485
Be Described by DFT-D. Journal of Chemical Theory and Computation.
486
2012;8(6):2034-43.
flavylium-(4→8)-flavan
chromophores.
Tetrahedron.
AC C
EP
TE D
M AN U
SC
and
RI PT
464
26
ACCEPTED MANUSCRIPT [27] Anouar EH, Gierschner J, Duroux J-L, Trouillas P. UV/Visible spectra of natural
488
polyphenols: A time-dependent density functional theory study. Food Chem.
489
2012;131(1):79-89.
490
[28] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR,
491
Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X,
492
Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M,
493
Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H,
494
Vreven T, Montgomery Jr. JA, Peralta JE, Ogliaro F, Bearpark MJ, Heyd J, Brothers
495
EN, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell
496
AP, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam NJ, Klene M, Knox JE,
497
Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O,
498
Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski
499
VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas Ö,
500
Foresman JB, Ortiz JV, Cioslowski J, Fox DJ. Gaussian 09. Wallingford, CT, USA:
501
Gaussian, Inc.; 2009.
502
[29] Vallverdu-Queralt A, Biler M, Meudec E, Guerneve CL, Vernhet A, Mazauric JP,
503
Legras JL, Loonis M, Trouillas P, Cheynier V, Dangles O. p-Hydroxyphenyl-
504
pyranoanthocyanins: An Experimental and Theoretical Investigation of Their Acid-Base
505
Properties and Molecular Interactions. Int J Mol Sci. 2016;17(11).
506
[30] Carvalho ARF, Oliveira J, de Freitas V, Mateus N, Melo A. Unusual Color Change
507
of Vinylpyranoanthocyanin-Phenolic Pigments. J Agric Food Chem. 2010;58(7):4292-
508
7.
509
[31] Carvalho ARF, Oliveira J, de Freitas V, Mateus N, Melo A. A theoretical
510
interpretation of the color of two classes of pyranoanthocyanins. Theochem-J Mol
511
Struct. 2010;948(1-3):61-4.
AC C
EP
TE D
M AN U
SC
RI PT
487
27
ACCEPTED MANUSCRIPT 512
[32] Anouar EH, Osman CP, Weber J-FF, Ismail NH. UV/Visible spectra of a series of
513
natural and synthesised anthraquinones: experimental and quantum chemical
514
approaches. SpringerPlus. 2014;3:233.
515
[33] Oliveira J, Mateus N, de Freitas V. Previous and recent advances in
516
pyranoanthocyanins
517
2014;100(0):190-200.
in
aqueous
solution.
518
Dyes
and
Pigments.
RI PT
equilibria
ACKNOWLEDGEMENTS
520
This work received financial support from FEDER funds through COMPETE,
521
POPH/FSE, QREN and FCT (Fundação para a Ciência e Tecnologia) by a post-doctoral
522
scholarship (SFRH/BPD/112465/2015), two investigator contracts (IF/00225/2015 and
523
IF/01355/2014) and grants PTDC/AGR-TEC/2789/2014, REDE/1517/RMN/2005. This
524
work
525
POCI/01/0145/FEDER/007265) from FCT/MEC through national funds and co-
526
financed by FEDER, under the Partnership Agreement PT2020.
M AN U support
(UID/QUI/50006/2013
-
TE D
financial
EP
528
received
AC C
527
also
SC
519
28
ACCEPTED MANUSCRIPT 529
Supplementary material
530
1.200
1.000
RI PT
0.143 mM 0.114 mM
0.600
0.086 mM 0.057 mM
0.400
0.200
0.000 250
350
450
550
650
λ (nm)
SC
Absorbance
0.800
750
850
Figure 8 – UV-Visible spectra of pigment 3 in aqueous solution (15% (v/v) ethanol) at pH 6 at
533
different molar concentrations from 0.057 to 0.143 mM.
M AN U
531 532
AC C
EP
TE D
534
29
ACCEPTED MANUSCRIPT 20.000
0.057 mM
0.086 mM
0.114 mM
0.143 mM
18.000 16.000
Absorbance
14.000 12.000 10.000
6.000 4.000 2.000 0.000 250
350
450
550
650
λ (nm)
750
RI PT
8.000
850
Figure 9 – Normalized UV-Visible spectra of pigment 3 in aqueous solution (15% (v/v) ethanol)
537
at pH 6 at different molar concentrations from 0.057 to 0.143 mM.
M AN U
SC
535 536
AC C
EP
TE D
538
30
ACCEPTED MANUSCRIPT 2.5
1.5
1
0.5
0 350
450
550
650
750
λ (nm) 0s
2179 s
7185 s
850
SC
250
RI PT
Absorbance
2
Figure 10 – Spectral changes observed for pigment 3 upon pH jumps from a stock solution of pH
541
1 in 0.1M HCl to pH 6.42 with time (0, 2179 and 7185 s).
M AN U
539 540
AC C
EP
TE D
542 543
31
ACCEPTED MANUSCRIPT
SC
RI PT
544
M AN U
545 546
Figure 4 – Theoretical (TD-DFT) UV-visible absorption spectra of dicationic (A), cationic (B), neutral
547
(C) and anionic (D) forms of pigments 1, 2 and 3.
AC C
EP
TE D
548
32
SC
RI PT
ACCEPTED MANUSCRIPT
549
Figure 5 – MO correlation diagram of dicationic (A), cationic (B), neutral (C) and anionic (D) forms of
551
pigment 1.
M AN U
550
AC C
EP
TE D
552
33
SC
RI PT
ACCEPTED MANUSCRIPT
553
Figure 6 – MO correlation diagram of dicationic (A), cationic (B), neutral (C) and anionic (D) forms of
555
pigment 2.
M AN U
554
AC C
EP
TE D
556
34
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 7 – MO correlation diagram of dicationic (A), cationic (B), neutral (C) and anionic (D) forms of
559
pigment 3.
AC C
EP
TE D
M AN U
557 558
35
ACCEPTED MANUSCRIPT •
Equilibrium forms of dimethylamino-based pyranoanthocyanins in aqueous solutions. The absence of hydration reactions was confirmed by UV-Visible spectroscopy.
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Structural features affected the protonation reaction at the amino group.
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Structural features affected the deprotonation in the pyranoflavylium moiety.
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