2′-Hydroxyflavylium: introducing flavanones into the flavylium network of chemical reactions

Article abstract of DOI:10.1016/j.tet.2007.11.007

Chalcones possessing a hydroxyl group in position 2 cyclize to form flavylium salts in acidic media, this reaction being reversible under neutral-basic conditions. On the other hand, chalcones possessing a hydroxyl group in position 2′ cyclize to form flavanones in basic media. By synthesizing 2′-hydroxyflavylium tetrafluoroborate, it was possible to obtain trans-2,2′-dihydroxychalcone that in solution can evolve to 2′-hydroxyflavanone or back to 2′-hydroxyflavylium depending on the pH. The several equilibria established in aqueous solution were fully characterized. The importance of including flavanones into the flavylium network of chemical reactions is briefly exploited.

Full text of DOI:10.1016/j.tet.2007.11.007

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 714e720
www.elsevier.com/locate/tet
20-Hydroxyflavylium: introducing flavanones into the flavylium
network of chemical reactions
*
Vesselin Petrov, Raquel Gomes, A. Jorge Parola , Alexandre Jesus,
*
´
Cesar A.T. Laia, Fernando Pina
REQUIMTE, Departamento de Qu´ımica, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa,
Campus da Caparica, 2829-516 Caparica, Portugal
Received 5 September 2007; received in revised form 25 October 2007; accepted 2 November 2007
Available online 6 November 2007
Abstract
Chalcones possessing a hydroxyl group in position 2 cyclize to form flavylium salts in acidic media, this reaction being reversible under
neutralebasic conditions. On the other hand, chalcones possessing a hydroxyl group in position 20 cyclize to form flavanones in basic media.
By synthesizing 20-hydroxyflavylium tetrafluoroborate, it was possible to obtain trans-2,20-dihydroxychalcone that in solution can evolve to
20-hydroxyflavanone or back to 20-hydroxyflavylium depending on the pH. The several equilibria established in aqueous solution were fully
characterized. The importance of including flavanones into the flavylium network of chemical reactions is briefly exploited.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
results from deprotonation of AHþ; the hemiketal species,
B, is obtained by hydration in the 2 position of the flavylium
cation; cis-2-hydroxychalcone, Cc, is an open form tautomer
of the hemiketal B, and trans-2-hydroxychalcone, Ct, results
from the cisetrans isomerization of Cc. In basic media, unpro-
tonated hemiketals and chalcones can be formed.
It is known that flavanones make part of the flavanoid bio-
synthesis pathways, being formed upon intramolecular cycli-
zation of chalcones through an enzymatic reaction involving
chalcone isomerase.4 On the other hand, they are biosynthetic
precursors of anthocyanins. Flavanones can also be syn-
thesized by non-enzymatic mechanisms from the adequate
chalcones.5 In general, these syntheses start from chalcones
lacking hydroxyl groups at position 2, thus preventing the in-
tramolecular formation of hemiketals and by consequence of
flavylium cations.6 On contrary, by using chalcones possessing
hydroxyl groups in both positions 2 and 20, they can either
cyclize to flavylium salts in acidic media or to flavanones in
moderately basic media. Herein, the extension of the flavylium
network of chemical reactions to the formation of flavanone
species is reported.
Chemical systems possessing two or more chemical species
that can be interconverted by external stimuli such as light, pH
variations, redox potential (multistate systems) are a matter of
great interest due to the possibility of conceiving switches,1
optical memories2 or even models that mimick the activity
of a neuron.3 In particular, the possibility of connecting two
or more switches in the same system is a step further to envis-
age logic gates at the molecular level.
The network of reversible chemical reactions originated by
flavylium ions in aqueous solutions is a paradigmatic example
of such kind of systems, Scheme 1. This general network is
also followed by anthocyanins, the molecules responsible for
the majority of red and blue colours of flowers and fruits.
As shown in Scheme 1, the flavylium cation, AHþ, is the
dominant species at low pH values. The quinoidal base, A,
* Corresponding authors. Tel.: þ351 212948355; fax: þ351 212948550.
E-mail addresses: ajp@dq.fct.unl.pt (A.J. Parola), fjp@dq.fct.unl.pt
(F. Pina).
0040-4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.11.007

V. Petrov et al. / Tetrahedron 64 (2008) 714e720
715
Scheme 1. Species originated by 20-hydroxyflavylium tetrafluoroborate in aqueous solution.
2. Results and discussion
20-Hydroxyflavanone was obtained from the flavylium ion,
by equilibrating a watereethanol (1:1) solution at pH ca. 9
followed by isolation of the compound as a precipitate. Full
assignment of the NMR spectra was possible on the basis of
COSY, HMQC and HMBC spectra. In particular, 60-H was
The synthesis of 20-hydroxyflavylium tetrafluoroborate was
accomplished by condensation of salicylaldehyde with 20-hy-
droxyacetophenone, following known methods.7 Full assign-
1
4
ment of the H NMR spectrum was made on the basis of the
identified in the COSY spectra through a J constant with
COSY spectrum, on the known fact that 4-H usually appears
as the lowest field signal in the NMR spectra of flavylium
salts,8 and that either 30-H or 50-H is expected to appear at
the highest field. These assignments were confirmed through a
NOESY spectrum where a cross peak between 3-H and 60-H
was observed. HMQC and HMBC experiments allowed full
assignment of the 13C NMR spectrum (see Supplementary data
for a list of HMBC connectivities).
trans-2,20-Dihydroxychalcone was obtained from the flavy-
lium salt by dissolution in 0.1 M NaOH, waiting for full con-
version to Ct2ꢀ and neutralization. The assignments of the
NMR signals were based on COSY, HMQC and HMBC spec-
tra. In particular, the connectivities of 60-H with 20-C and with
the carbonyl carbon in the HMBC spectrum unequivocally
identify 60-H, and thereafter it is straightforward to assign
the other ring B protons using the COSY spectrum. Similarly,
the connectivity between 6-H and b-C in the HMBC spectrum
leads to full assignment of ring A protons. An interesting fea-
ture in the 1H NMR spectrum of this chalcone is the chemical
shifts of the two hydroxyl protons, appearing at d 12.9 and
5.9 ppm. The low field of the former and the large difference
between both signals indicate that the hydroxyl group in posi-
tion 20 is H-bonded to the carbonyl group. This suggests that
the hydroxyl group in position 2 is more acidic, as presented
in Scheme 1.
2-H, and 5-H is recognized in the HMBC spectra by its
connectivity with 4-C of the carbonyl group.
When dissolved in aqueous solution, 20-hydroxyflavylium
tetrafluoroborate gives rise to the network of chemical reac-
tions shown in Scheme 1. Most of the network of 20-hydroxy-
flavylium is similar to those of other flavylium compounds.1a
The flavylium ion, AHþ, can either loose a proton to the
solvent to form the quinoidal base A or be hydrated to form
hemiketal B; the hemiketal can tautomerize to cis-chalcone,
Cc and the latter can isomerize to the trans-chalcone, Ct. At
basic pH values, all species can deprotonate to form anionic
species like the ionized chalcones in Scheme 1. The novelty
of this flavylium cation is conferred by the position of a hy-
droxyl in position 20, which in a narrow pH range gives rise
to the formation of 20-hydroxyflavanone, F.
In acidic media, the network of chemical reactions reported
in Scheme 1 of the manuscript can be accounted for by the
following set of equations:
AHþ þ H2O#A þ H3Oþ Ka
ð1Þ
AHþ þ 2H2O#B þ H3Oþ Kh
B#Cc Kt
ð2Þ
ð3Þ

716
V. Petrov et al. / Tetrahedron 64 (2008) 714e720
A
B
C
0.8
0.6
0.4
0.2
0
0.7
pK =4.8
a
^
0.5
pK =3.15
0.6
'
a
a
pK =2.6
0.8
0.4
0
0.8
0
1 2 3 4 5 6 7 8
pH
0
0 1
2
3
4
5
6
0
0.4
pH
0
1
2
3
4
5
6
7
pH
0
400
500
Wavelength/nm
600
200
300
400
500
600
700
200
300
400
500
600
Wavelength/nm
Wavelength/nm
Figure 1. (A) Equilibrated aqueous solutions of compound 20-hydroxyflavylium tetrafluoroborate (after 1 day) as a function of pH; (B) the same after 1 min; (C)
stopped flow data taken after 20 ms.
flow, and its rate constant is slightly pH dependent (1.5 sꢀ1
at pH¼3; 1.0 sꢀ1 at pH¼9). In Figure 1C, the absorption spec-
tra as a function of pH taken after 20 ms are presented, allow-
ing to calculate Ka¼10ꢀ4.8. The pKas of the chalcones could
also be determined upon pH jumps from acidic solutions of
AHþ to the neutralebasic region (cis-chalcones) and from
basic solutions of Ct2ꢀ to the neutralebasic region (trans-
chalcones), see Supplementary data.
Taking profit from the fact that the cisetrans isomerization
is very slow (several hours), making the pseudo-equilibrium
long lived, the following experiment was carried out: after
a pH jump from 1 to 6.3 to form the pseudo-equilibrium,
the solution was introduced in the stopped flow and a ‘reverse’
pH jump back to acidic values was monitored, see Figure 2.
Figure 2 is compatible with two consecutive kinetic pro-
cesses. The first one is very fast and occurs within the mixing
time of the stopped flow experiments. At pH¼6.3, the system
Cc#Ct Ki
ð4Þ
Eqs. 1e4 can be substituted by a single acidebase equili-
brium,2a as shown in Eq. 5:
AHþ þ 2H2O#CB þ H3Oþ Ka0
ð5Þ
ð6Þ
ð7Þ
where
½CBꢁ ¼ ½Aꢁ þ ½Bꢁ þ ½Ccꢁ þ ½Ctꢁ
and
Ka0 ¼ Ka þ Kh þ KhKt þ KhKtKi
The pH dependent spectral variations of equilibrated solu-
tions of compound 20-hydroxyflavylium are reported in Fig-
ure 1A. The flavylium cation, AHþ, is the dominant species
at low pH values, while at higher pH values, Ct dominates
the equilibrium composition. From the data reported in Fig-
0
ure 1A, the global constant Ka ¼KaþKhþKhKtþKhKtKi¼
10ꢀ2.6 is obtained.
0.25
In the cases, as in the present compound, where an activa-
tion barrier exists for the interconversion of cis- to trans-
chalcone, a pseudo-equilibrium can also be defined:
pH=1.34
0.2
AHþ þ 2H2O#ðA þ Cc þ BÞ þ H3Oþ K^a
ð8Þ
As in Eq. 5, the pseudo-equilibrium can be accounted for by
an acidebase reaction, Eq. 8, where
0.15
0.1
0.05
0
pH=2.09
pH=2.73
K^a ¼ Ka þ Kh þ KhKt
ð9Þ
pH=3.54
There is experimental evidence (see below) that the pseudo-
equilibrium of the present compound is constituted by Cc
and B (major forms) together with a very low percentage of
A. Figure 1B represents the pseudo-equilibrium, from which
an observed constant, K^a¼KaþKhþKhKt¼10ꢀ3.15 can be
calculated.
When a pH jump from the stock solutions at pH¼1.0 to
higher pH values is carried out, the first reaction to occur is
the formation of the base, A, followed by the appearance of
the pseudo-equilibrium and finally the ‘real’ equilibrium.
The disappearance of the base, A, was monitored by stopped
pH=4.37
1.6
0.4
0.8
1.2
2
Time/s
Figure 2. Stopped flow traces corresponding to reverse pH jumps carried out
from pH¼6.3 to the indicated pH (upon previous pH jump from pH¼1 to
pH¼6.3).

V. Petrov et al. / Tetrahedron 64 (2008) 714e720
717
is mainly constituted by B and Cc in the pseudo-equilibrium,
and by consequence the first kinetic process can only be attrib-
uted to the (very fast) formation of AHþ at the expenses of B,
through the hydrationedehydration mechanism. The slowest
second kinetic process (responsible for the traces in Fig. 2)
exhibits a rate constant of 3.0ꢂ0.2 sꢀ1 and is practically pH
independent. It can be attributed to the formation of AHþ
from Cc, involving a tautomerization process through B.
Moreover, the amplitudes of the two processes should be
proportional to the concentrations of Cc and B at the
pseudo-equilibrium, allowing to calculate Kt¼0.46. Using
Eqs. 7 and 9 and the values of Ka and Kt, Kh¼4.8ꢃ10ꢀ4 and
Ki¼8.2 can be obtained.
and only at pH>10 leads to Ct2ꢀ, suggesting the involvement
of mono-ionized flavanone, Fꢀ, in this reaction.
The trans-chalconeeflavanone equilibrium has received
large attention.9 It has been established that (i) both species
are stable in the acidic and neutral region; (ii) they start to in-
terconvert under basic conditions; (iii) as the pH increases, the
intermediate of the cyclization and ring-opening reactions
shifts progressively from an enolate to a carbanion intermedi-
ate mechanism.9 Our observations clearly support the need of
ionized species for the interconversion to occur. The chalcone
starts to form flavanone only when Ctꢀ is present (pKCt1¼8.3)
while conversion of flavanone to chalcone starts at pHz10,
a value expected for the F/Fꢀ pKa.9d This peculiar situation,
where flavanone is formed from Ctꢀ at lower pH values
(from ca. pH>7) and leads to Ct2ꢀ at higher pH values, is
responsible for the formation of a pH dependent equilibrium
involving F and Ct2ꢀ whose inflection point occurs at
pH¼11.7, see Figure 4.
2.1. Flavanone equilibrium
When an aqueous solution of flavylium cation at pH¼1.0 is
subjected to a pH jump to 8.25, Figure 3, the observed spectral
changes are compatible with two consecutive first order ki-
netic processes. The first step is the trivial formation of ionized
The thermodynamic data reported above allows to calculate
the mole fraction distribution of the different species as a func-
tion of pH. A thermodynamic equilibrium can be reached
starting either from AHþ at pH¼1 or from Ct2ꢀ at pH¼12.
In the first case, the quinoidal base A is the first species to
be formed, Figure 5A, but rapidly leads to the pseudo-equilib-
rium described in Figure 5B; the final equilibrium involves
AHþ and Ct in the acidic region and F and Ct2ꢀ in the basic
region, Figure 5C. Starting from Ct2ꢀ at pH¼12, the final
equilibrium is the same (Fig. 5C) but the pseudo-equilibrium
is different (Fig. 5D), containing unprotonated trans-chalcones
instead of their cis-isomers.
trans-chalcones (at this pH, Ctꢀ is in equilibrium with Ct2ꢀ
;
see Supplementary data) with kobs1¼0.1 minꢀ1; the second
one corresponds to the formation of flavanone F in equilibrium
with Ctꢀ/Ct2ꢀ, occurring with kobs2¼0.011 minꢀ1
.
Pure 20-hydroxyflavanone was isolated by precipitation
from saturated solutions of this mixture and characterized by
NMR, elemental analysis and MS, allowing to confirm the
spectral evolution of trans-chalconate towards flavanone for-
mation. The species Ctꢀ is clearly the immediate precursor
for the formation of flavanone, which is neither formed at
lower pH values (from Ct) nor at higher pH values (from
Ct2ꢀ). On the other hand, flavanone is stable at low pH values
On the other hand, starting from flavanone at neutral pH
leads to a different thermodynamic state, where only F and
Ct2ꢀ are present at the final equilibrium (Fig. 5E), since the
flavanone is stable in the acidic region.
0.15
-1
k
=0.1 min
obs1
obs2
-1
k
=0.011 min
0.8
pKap=11.7
0.4
0.2
0
0.8
0
0
0
50 100 150 200 250 300
Time/min
6
7
8
9
10 11 12 13
pH
0.4
200
300
400
500
600
Wavelength/nm
0
200
300
400
Wavelength/nm
500
600
Figure 3. Spectral modifications upon a pH jump of 4.0ꢃ10ꢀ5 M 20-hydroxy-
flavylium, from 1 to 8.25, showing formation of ionized trans-chalcones (Ctꢀ/
Ct2ꢀ) followed by the appearance of flavanone (full lines); spectrum of pure
flavanone (traced line).
Figure 4. Titration of 4.4ꢃ10ꢀ5 M flavanone towards basic pH values, with
formation of Ct2ꢀ
.

718
V. Petrov et al. / Tetrahedron 64 (2008) 714e720
1
A
1
B
AH+
Cc2-
AH+
A
Cc-
B
Cc
A
0
0
0
2
4
6
8
10
12
0
2
4
6
8
10
12
pH
pH
1
E
1
1
D
C
AH+
Ct2-
Ct
AH+
Ct2-
Ct
F
F
Ct2-
Ct-
0
0
0
2
4
6
8
10
12
2
4
6
8
10
12
2
4
6
8
10
12
pH
pH
pH
Figure 5. Mole fraction distribution of the species upon pH jumps from AHþ at pH¼1 to higher pH values (Adafter 25 ms, Bdpseudo-equilibrium after 1 min,
Cdfinal equilibrium after 24 h), from Ct2ꢀ at pH¼12 to lower pH values (Ddpseudo-equilibrium after 1 min) and from F at neutral pH values (Edfinal equi-
librium after ca. 1 min).
Figure 6 illustrates the integration of the flavanone into the
flavylium network of chemical reactions. Starting from AHþ
at pH¼1 and carrying out a pH jump to 12, the ionized cis-
chalcone in equilibrium with B/Bꢀ is immediately formed.
This reaction was followed by stopped flow and occurs with
a rate constant of 1.8 sꢀ1. The thermodynamic equilibrium,
with only Ct2ꢀ, is reached upon ca. 3 h at pH¼12, Figure 6A.
As can be seen in Figure 6B, a second pH jump to 8.5 gives
rise to the formation of flavanone. The flavanone at acidic
pH values is stable, but at pH¼12 leads to Ct2ꢀ in the time
scale of seconds. The recovery of the initial Ct2ꢀ species is
practically complete, see Figure 6C.
4. Experimental
4.1. Synthesis
All reagents and solvents used were of analytical grade.
NMR spectra were run on a Bruker AMX 400 instrument op-
erating at 400.13 MHz (1H) and 100.00 MHz (13C). COSY,
HMQC, HMBC and eventually NOESY spectra were run on
each sample to allow full assignment of the NMR peaks. Field
Desorption MS spectra were run on a Micromass GCT
machine and elemental analysis was obtained on a Thermo-
finnigan Flash EA 1112 Series instrument.
The chalconeeflavanone equilibrium in the present system
does not exhibit photochemistry. However, some flavanones
can be photochemically converted into chalcones in organic
solvents.10 This possibility is being explored in the framework
of multistate flavylium networks.
4.1.1. 20-Hydroxyflavylium tetrafluoroborate
3'
2'
HO
4'
+
O
B
8
5'
7
6
6'
A
C
3
4
5
3. Conclusions
20-Hydroxyflavylium tetrafluoroborate was prepared according
to a procedure adapted from Katritzky et al.7 Salicylaldehyde
(1.1 ml, 10 mmol) and 20-hydroxyacetophenone (1.2 ml,
10 mmol) were dissolved in 10 ml of acetic acid and 2 ml of
HBF4. Acetic anhydride (8.5 ml) was then added dropwise
and the temperature of the reaction mixture rose until 75 ꢄC.
The reaction mixture became red and was stirred overnight.
A yellow precipitate had formed that was filtered off, washed
with water and then carefully with diethyl ether and dried
The introduction of two hydroxyl groups in positions 2 and
20 of trans-chalcone defines a multistate system that responds
to pH changes by evolving to a cis-chalcone through an isom-
erization or to a flavanone through a cyclization process. The
multiequilibria were fully characterized and show that flava-
nones may be used to enlarge the number of states in flavylium
based multistateemultifunctional systems.

V. Petrov et al. / Tetrahedron 64 (2008) 714e720
719
0.8
B
0.8
0.6
0.4
0.2
0
A
pH jump 12 to 8.5
Ct-
Flavanone
pH jump 1 to 12
1.8s-1
Cc2-/B-
Ct2-
AH+
0.6
0.4
0.2
0
equil. (3 hours)
initial
initial
Cc2-/B-
equil.
250 300 350 400 450 500 550 600
Wavelength/nm
250 300 350 400 450 500 550 600
Wavelength/nm
D
0.8
0.6
0.4
0.2
0
C
pH jump 8.5 to 12
very fast
Ct2-
Flavanone
pH=1
pH=1
pH=8.5
pH=12
AH+
stable
Ct2-
stable
-
Ct
Ct
pH=12
pH=1
Flavanone
stable
pH=8.5
Flavanone
stable
pH=8.5
250 300 350 400 450 500 550 600
Wavelength/nm
Figure 6. (A) Spectral modifications upon a pH jump of 4.0ꢃ10ꢀ5 M 20-hydroxyflavylium, from 1 to 12; (B) the same upon a pH jump form equilibrated solutions
at pH¼12 to 8.5. The flavanone is formed during this step; (C) the spectrum obtained from the flavanone upon a pH jump to 12 (traced line) is the same of the
initial species (full line); (D) diagram showing how the flavanone can be integrated in the network of the flavylium.
(0.48 g, 16.3%). 1H NMRy (400.13 MHz, CDCl3) dH 9.31 (1H,
d, J 9.21, 4-H), 9.13 (1H, d, J 9.2, 3-H), 8.27 (1H, m, 60-H),
8.23 (1H, m, 5-H), 8.19 (1H, m, 7-H), 8.16 (1H, m, 8-H),
(1H, t, J 7.7, 40-H), 7.03 (1H, d, J 8.4, 3-H), 7.00 (1H, t,
J 7.7, 5-H), 6.94 (1H, t, J 7.7, 50-H), 6.87 (1H, d, J 8.1, 30-H),
5.89 (1H, br s, 2-OH). 13C NMR (100.00 MHz, CDCl3) dC
194.5 (C carbonyl), 163.6 (20-C), 155.6 (2-C), 141.5 (b-C),
136.3 (4-C), 132.1 (40-C), 130.1 (6-C), 130.0 (10-C), 129.9
(60-C), 121.3 (1-C), 121.2 (5-C or a-C), 118.9 (5-C or a-C),
118.6 (50-C), 116.8 (3-C), 116.6 (30-C). FD-MS m/z: 240.08
[M]þ (100%). EA Found: C, 74.46; H, 5.03. Calcd for
C15H12O3: C, 74.99; H, 5.03.
4
7.94 (1H, t, J 7.4, 6-H), 7.71 (1H, td, J 7.6, J 1.5, 40-H),
7.33 (1H, d, J 8.5, 30-H), 7.16 (1H, t, J 7.6, 50-H). 13C NMR
(100.00 MHz, CDCl3) dC 175.0 (2-C), 163.7 (20-C), 155.4
(8a-C), 141.4 (40-C), 138.8 (7-C), 130.4 (60-C), 130.2 (5-C
and 6-C), 123.7 (4a-C), 122.3 (40-C), 122.1 (50-C), 119.9 (30-
C), 118.9 (8-C), 114.6 (10-C), 113.2 (3-C). FD-MS m/z:
222.06 [MꢀH]þ (100%). EA Found: C, 58.52; H, 3.79. Calcd
for C15H11BF4O2: C, 58.11; H, 3.58.
4.1.3. 20-Hydroxyflavanone
3'
HO
4'
4.1.2. trans-2,20-Dihydroxychalcone
2'
1'
8
O
5'
7
6
2
6'
3
3'
6'
1
HO
OH
α
2
4'
5'
4
5
4
3
2'
5
O
1
1'
20-Hydroxyflavylium tetrafluoroborate was dissolved in a mix-
ture of water and ethanol (1:1) and the pH was adjusted to 9.
By the following day, ethanol had evaporated and a white solid
precipitated from the aqueous solution. 20-Hydroxyflavanone
was then filtered off, carefully washed with water and dried.
It was recrystallized from a solvent mixture of chloroform
and hexane. 1H NMR (400.13 MHz, CDCl3) dH 7.97 (1H,
dd, J 7.8, J 1.6, 5-H), 7.54 (1H, td, J 7.5, J 1.6, 7-H), 7.32
(1H, d, J 7.6, 60-H), 7.27 (1H, t, J 7.6, 40-H), 7.10 (1H, t,
J 7.6, 6-H), 7.09 (1H, d, J 7.5, 8-H), 6.99 (1H, t, J 7.6, 50-H),
6.92 (1H, d, J 8.1, 30-H), 6.62 (1H, br s, 20-OH), 5.78 (1H,
6
β
O
20-Hydroxyflavylium tetrafluoroborate was dissolved in water
at pH¼13 and allowed to equilibrate. The solution was then
carefully acidified to pH¼7. After a few moments in an ice
bath, trans-2,20-dihydroxychalcone precipitated. The yellow
solid was filtered and washed with cold water. 1H NMR
(400.13 MHz, CDCl3) dH 12.92 (1H, s, 20-OH), 8.22 (1H, d,
J 15.6, b-H), 8.06 (1H, d, J 8.0, 60-H), 7.98 (1H, d, J 15.6,
a-H), 7.69 (1H, d, J 7.6, 6-H), 7.50 (1H, t, J 8.0, 4-H), 7.30
4
4
y
Values for chemical shifts are presented in parts per million and coupling
constants in Hertz. Unless otherwise noted, coupling constants are vicinal.
2
dd, J 13.0, 3.0, 2-H), 3.16 (1H, dd, J 13.0, J 17.1, 3a-H),

720
V. Petrov et al. / Tetrahedron 64 (2008) 714e720
2
3.01 (1H, dd, J 3.0, J 17.1, 3b-H). 13C NMR (100.00 MHz,
CDCl3) dC 192.9 (4-C), 161.3 (8a-C), 153.5 (20-C), 136.4
(7-C), 129.8 (40-C), 127.3 (5-C), 126.8 (60-C), 124.6 (10-C),
122.1 (6-C), 121.1 (4a-C), 120.8 (50-C), 118.1 (8-C), 116.þ6
(30-C), 77.2 (2-C), 43.0 (3-C). FD-MS m/z: 240.08 [M]
(100%). EA Found: C, 73.53; H, 5.36. Calcd for
C15H12O3$1/3H2O: C, 73.16; H, 5.18.
Supplementary data
Supplementary data associated with this article can be
found in the online version, at doi:10.1016/j.tet.2007.11.007.
References and notes
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4.2. General
All experiments were carried out in aqueous solution. The
pH was adjusted by addition of HCl and NaOH, or buffer, and
was measured in a Meterlab pHM240 pH meter from Radio-
meter Copenhagen. UV/vis absorption spectra were recorded
in a Shimadzu UV2501-PC spectrophotometer.
pH jumps were done by adding a certain volume of a stock
solution of the flavylium salt in 0.1 M HCl to a 10 cm3 flask
containing an equivalent amount of NaOH to neutralize
HCl and 1 cm3 of 0.1 M universal buffer of Theorell and
Stenhagen11 at the desired final pH followed by the addition
of water to the final volume. The spectra were measured
immediately (ca. 1 min) and the solutions kept in the dark to
follow the establishment of the final equilibrium. Reverse
pH jumps involved a second pH jump to a lower pH value,
by addition of HCl.
The titrations of flavanone and of trans-chalcone were
made in a batch process due to their reactivity. An aqueous so-
lution containing 0.1 M buffer was actually titrated, when the
desired pH was reached, 2 ml of the solution was transferred to
a dry cuvette and an aliquot of the respective stock solution
added (25 ml of flavanone in EtOH, 5 ml of Ct2ꢀ at pH¼12).
The spectrum was run immediately and the pH controlled at
the end.
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previously.12
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´
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Acknowledgements
12th ed.; Hoepli: Milano, 1982; pp 157e160. This universal buffer is
prepared in the following way: dissolve 2.3 cm3 of phosphoric acid
(85% (w/w)), 7.00 g of mono-hydrated citric acid and 3.54 g of boric
acid in some water, add 343 ml of 1 M NaOH and dilute with water until
1 dm3.
Portuguese FCT-MCTES under Project no. POCI/QUI/
57735/2004, and grants SFRH/BPD/18214/2004 (V.P.) and
SFRH/BD/27282/2006 (R.G.) are acknowledged for financial
support.
12. Maestri, M.; Ballardini, R.; Pina, F.; Melo, M. J. J. Chem. Educ. 1997, 74,
1314e1316.