Unveiling the chemistry behind bromination of quercetin: The 'violet chromogen'

Article abstract of DOI:10.1016/j.tetlet.2014.01.081

Bromination of quercetin with N-bromosuccinimide in neutral aqueous methanol occurs surprisingly in the electron-deficient A-ring only. Deprotonation of the acidic 7-OH is a major driver of this regioselective reaction. The increase of electron density ma

Full text of DOI:10.1016/j.tetlet.2014.01.081

Tetrahedron Letters 55 (2014) 1602–1607
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Tetrahedron Letters
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Unveiling the chemistry behind bromination of quercetin: the ‘violet
chromogen’
⇑
Mario C. Foti , Concetta Rocco
Istituto di Chimica Biomolecolare del CNR, via P. Gaifami 18, I-95126 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Bromination of quercetin with N-bromosuccinimide in neutral aqueous methanol occurs surprisingly in
the electron-deficient A-ring only. Deprotonation of the acidic 7-OH is a major driver of this regioselective
reaction. The increase of electron density makes in fact the quercetin anion suitable for an electrophilic
attack by bromine at positions 8 and 6. Several pieces of evidence (NMR spectra and H/D exchange) are
presented to substantiate the mechanism advanced. Bromoquinones/quinomethides produced in excess
of N-bromosuccinimide are responsible for the formation of a stable ‘violet chromogen’.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 13 December 2013
Revised 15 January 2014
Accepted 20 January 2014
Available online 29 January 2014
Keywords:
Quercetin
Bromination
Deprotonation
Violet chromogen
Quinones
Quercetin (1) is
a
polyphenol belonging to the class of
and biology of quercetin is therefore notable, and in the last dec-
ades has shown no decline.8
‘flavonoids’ which are widely distributed in the plant kingdom
and consequently in our daily diet.1,2 Quercetin has recognized bio-
logical properties3–5 and—as most phenols6—is able to slow down
the process of oxidation of organic matter7 caused by dioxygen
3O2 (peroxidation). This beneficial antioxidant property is due to
the ability that quercetin has to chelate transition-metal ions and
to quench peroxyl radicals ROOÅ.6,7 The interest in the chemistry
The oxidation chemistry of quercetin has long been investi-
gated.9–11 The two-electron oxidation yields quinone/quinomet-
hide compounds (Eq. (1)) that are intensely colored in purple
(kmax ꢀ 525 nm, in 80% by volume methanol/water).9 Density func-
tional theory (DFT) calculations12 show that the quinomethide Q1
is more stable—and thus more abundant in solution—than the
other three possible tautomers (Eq. (1)).
H
O
O
O
HO
O
O
H
HO
O
O
H
+
O
free-radicals
O
H
MeOH/H2O
O
O
O
H
H
O
O
O
O
ð1Þ
1
( )
(Q1)
HO
O
HO
O
O
O
OH
OH
+
+
+
OH
O
O
OH OH
OH
O
O
OH
Decreasing stability
⇑
Corresponding author. Tel.: +39 095 733 8343; fax: +39 095 733 8310.
E-mail address: mario.foti@cnr.it (M.C. Foti).
http://dx.doi.org/10.1016/j.tetlet.2014.01.081
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

M. C. Foti, C. Rocco / Tetrahedron Letters 55 (2014) 1602–1607
1603
In protic solvents, the survival of the above quinone/quinomet-
hide species (reaction 1) is however very limited. In fact, in 80%
methanol/water (v/v) the half-life of Q1 is 2.5 s only.9 Q1 can be
regarded as a resonance-stabilized benzylic carbocation which
readily undergoes a proton-assisted (Michael-type) nucleophilic
addition of solvent (ROH) at position 2 (and 3) (Eq. (2)).13,14 Discol-
oration of the solution follows this reaction as a consequence of the
interruption of the conjugation between the rings B and A+C (Eq.
(2)). At room temperature, the reaction in 80% methanol/water
(v/v) is over, that is the purple color disappears, in a few tens of
seconds.9
sodium, and ceftriaxone sodium) with a simple and accurate spec-
trophotometric test.16,17
The authors of these works attributed the violet color to the
formation of quercetin quinones/quinomethides, in particular
to Q1.15,16 This hypothesis has been reconfirmed until recently16
after about 20 years from the first observation. Our data, how-
ever, do not support this conclusion because Q1 disappears very
quickly in methanol/water mixtures (see above). What is (are)
therefore the compound(s) responsible for this persistent and
intense violet color? While answering this question we chanced
upon a few derivatives of quercetin (bromoquercetins and
H
O
H
O
O
OH
OR
HO
O
O
HO
O
O
ð2Þ
ROH
1,6-addition
O
O
OH
OH
The aforementioned instability of Q1 in protic solvents, how-
20-hydroxy-6,8-dibromoquercetin, see Scheme 1) worthy
of being mentioned because we discovered they possess
singular properties that will be reported in a forthcoming Letter.
Although a few of these compounds are already known,18 the
syntheses we now report (see Supplementary data) are particu-
larly simple and environmentally-friendly deserving therefore
consideration.
First, we verified that upon treatment of a methanol solution of
quercetin with aqueous NBS in a mole ratio of 1:4, respectively, the
solution became immediately violet and the color persisted for
ever, seems to contrast with a report of 1992 in which the authors
affirm that a methanolic solution of quercetin upon treatment with
a neutral aqueous solution of N-bromosuccinimide (NBS) produced
instantaneously an intense ‘violet chromogen’ (kmax ꢀ 510 nm)
which was stable for at least 15 min.15 Later, the procedure was
slightly modified and it was reported that the violet color persisted
without decaying for more than one hour.16,17 Interestingly, solu-
tions of this oxidized quercetin reagent were used to titrate ascor-
bic acid and several antibiotics (cefoperazone sodium, cefazolin
OH
OH
OH
OH
OH
Br
Br
HO
Br
O
HO
Br
O
HO
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
(2)
(3)
(4)
OAc
OH
OAc
AcO
O
OAc
HO
AcO
OAc
Br
Br
AcO
Br
HO
Br
O
AcO
Br
O
OAc
OH
OAc
OAc
O
OH
O
OAc
O
(5)
(6)
(7)
OAc
AcO
OAc
AcO
O
OAc
OAc
O
(8)
Scheme 1. Quercetin bromoderivatives obtained by treating a methanol solution of quercetin with aqueous NBS followed by reduction with Na2S2O4 at room temperature.
The acetates were obtained by treating the reaction mixtures with acetic anhydride/pyridine.

1604
M. C. Foti, C. Rocco / Tetrahedron Letters 55 (2014) 1602–1607
Figure 1. (1) NMR spectrum of quercetin 1 (19.2 mM) in methanol-d4; the peaks a, b, and c correspond to the B-ring protons 20, 60, and 50, respectively; d and e correspond to
the A-ring protons 8 and 6, respectively. (2) Addition of a D2O-solution of NBS (91.2 mM) at a mole ratio NBS:1 of 0.5; the peaks a0, b0, c0, and e0 correspond to the protons 20, 60,
50, and 6, respectively, of 8-bromoquercetin 4. (3) Same as for (2) with NBS:1 of 0.85. (4) NBS:1 of 1.6. (5) NBS:1 of 2.8. (6) NBS:1 of 3.8.
hours. The presence of water was essential since in pure methanol
there was no formation of the chromogen. The reaction in deuter-
ated methanol+D2O had the same outcome but the 1H NMR spec-
trum of the solution—recorded immediately after mixing the
chemicals—was difficult to interpret because it contained six weak
and partially unresolved groups of signals scattered in the chemi-
cal shift range 5.8–8.5 ppm (see Fig. 1). The intensity of the signals
decreased in time and after about 15 min the signals disappeared
from the NMR spectra. We believe this peculiar behavior of the
proton signals was due to free-radicals in solution which broad-
ened the signals beyond detection (paramagnetic relaxation
enhancement).19,20
In previous experiments on the kinetics of oxidation of quer-
cetin with dpphÅ,9 it was observed that the quinomethide Q1
could be reconverted to quercetin (in non-protic solvents, e.g.,
CH2Cl2) by treatment with excess ascorbic acid palmitate at
room temperature (see the Supporting information of Ref. 9).
The UV–vis spectra showed a complete and ‘clean’ formation
of quercetin and disappearance of the colored quinone
(kmax ꢁ 525 nm) with the maintenance of an isosbestic point at
about 392 nm. We therefore treated a methanol/water solution
of the violet chromogen—obtained as described above—with
ascorbic acid or (better) sodium dithionite Na2S2O4 which caused
a quick discoloration of the solution and the formation of a pre-
cipitate. The NMR and MS spectra revealed that the precipitate
was 6,8-dibromoquercetin 2 (97% pure). The final yield of this
one-pot synthesis was about 70%. By changing the pH of the
solution we selectively obtained the 6-bromoquercetin 3 (adding
NaOH) and the 8-bromoquercetin 4 (adding HCl) in mixture with
variable quantities of 2. Upon changing the solvent from aqueous
methanol to aqueous acetone the reaction yielded reproducible
and significant quantities of a new bromoflavonoid 5 together
with 6,8-dibromoquercetin 2 in a 1:1 ratio (see Supplementary
data). The precipitate, obtained after treatment with Na2S2O4,
was acetylated with acetic anhydride/pyridine in CH2Cl2 at reflux
and chromatographed over silica gel to yield the acetylated form
6
(final yield 35%) and small quantities of
7 and 8 (see
Scheme 1).21
The foregoing data suggest that in neutral or weekly acidic solu-
tions the ‘violet chromogen’ is principally constituted of a mixture
of 6,8-dibromo- and 8-bromoquinones/quinomethides in tauto-
meric equilibrium with their respective isomers (Eq. (3)). These
are easily reduced by Na2S2O4 to brominated quercetin (Eq. (4)).
The 1H NMR spectrum of the ‘violet chromogen’, although weak
(see above), is compatible with the structure of the above
compounds.

M. C. Foti, C. Rocco / Tetrahedron Letters 55 (2014) 1602–1607
1605
OH
OH
O
Br
HO
O
O
HO
X
O
O
OH
NBS
ð3Þ
other
+ tautomers
O
MeOH/H2O
OH
OH
OH
O
Br
OH
Br
HO
X
O
O
OH
HO
X
O
other
OH
Na2S2O4
ð4Þ
+ tautomers
O
MeOH/H2O
OH
OH
OH
O
X = H; Br
The presence of bromine in the A-ring makes the quinones/qui-
In fact, the NMR spectra of 1 added with scalar amounts of NBS
depicted a different scenario, see Figure 1. Addition of increasing
amounts of NBS in D2O to a methanol-d4 solution of 1 caused
nomethides survive in protic solvents almost indefinitely. The
same is not true for the non-brominated counterparts produced
in (Eq. (1)) which react quickly with protic solvents (Eq. (2)).9 It
is likely that bromine destabilizes the benzylic carbocation men-
tioned above (Eq. (2)) by field effects. In other words, we think that
the zwitterionic forms I and II are less important than the covalent
form III in the resonance hybrid (see Scheme 2) of the brominated
quinones/quinomethides. This might therefore make the addition
of ROH more difficult.
Further, it is worth noting that under our experimental condi-
tions bromination of quercetin occurred in the A-ring only—
although the apparent target of this electrophilic aromatic substi-
tution should be the B-ring instead. The catechol ring of quercetin
is, in fact, electron-richer than the A-ring which suffers the with-
drawing field and mesomeric effects of the carbonyl group at the
4-position.9–11,22,23
To explain the regioselectivity of NBS, we had initially ad-
vanced the hypothesis that quercetin was rapidly oxidized to
the quinone form Q1 by NBS rather than being brominated. Oxi-
dation of quercetin in aqueous media is expected to be fast be-
cause it can involve an electron-transfer process from quercetin
anions (vide infra).9 The A-ring of oxidized quercetin (Q1) is elec-
tron richer than the B-ring and could therefore be the accessible
site for bromination. However, this explanation demanded
another restrictive requirement. Bromination of Q1 had to occur
faster than its decay by solvent addition (Eq. (2)). The fact that
reaction 2 is fast (vide supra) and that Q1 is a deactivated
substrate for electrophilic reactions makes this requirement
difficult to meet.
exclusively the formation of monobromoquercetin
4 and of
dibromoquercetin 2 (at higher ratios NBS:1, see Fig. 1) without
involving Q1 or any oxidized forms of bromoquercetins. In other
words, bromination occurred directly onto the A-ring of quercetin.
Quinones were formed after bromination when the molar ratio
NBS:1 exceeded 3. This finding forced us to abandon the explana-
tion given above and to assess the effects of the hydroxyls of the
A-ring on the reaction.
The 1H NMR spectrum of quercetin in acetone-d6 displays five
sharp peaks attributed to the five OHs by long-range experiments,
see Figure 2. Addition of H2O (or traces of NaOD) caused a large
broadening or disappearance (H2O >8% by volume) of the signal
of the 7-OH (d 9.75), see Figure 2. This suggests that the acidity
of the 7-OH is higher than that of the other OHs.24 Hence, the
7-OH must be the primary site of deprotonation of quercetin. The
behavior of the C-7, H-6, and H-8 NMR signals after the addition
of NaOD reinforces this conclusion.25 Indeed, our findings are in
agreement with the results of several other investigations which
all support the conclusion that the 7-OH is the most acidic hydro-
xyl in quercetin.26 In particular, Litwinienko and co-workers26 have
recently estimated that the pKa of the 7-OH is in the range 7.5–8.5.
They have also analyzed the implications that this relatively large
acidity has in the reactions of quercetin with radicals in ionizing
solvents.
On the basis of the pKa range reported above, it is plausible that
the anion of quercetin, as for other phenols,27 may well be the true
substrate of bromination, see Scheme 3. That is, it is most likely that
O
O
O
O
O
Br
Br
Br
HO
Br
O
H
HO
Br
O
H
HO
Br
O
H
O
O
O
O
OH
O
OH
O
OH
O
(II)
(III)
(I)
Scheme 2. Canonical resonance structures of dibromoquercetin quinomethide.

1606
M. C. Foti, C. Rocco / Tetrahedron Letters 55 (2014) 1602–1607
Figure 2. (1) 1H NMR of quercetin in acetone-d6 showing the region of the OH resonances. The assignment was done by HMBC experiments. (2) Same as above after the
addition of 4% (v/v) H2O. The hump at about 10.7 ppm is due to the 7-OH which is the most acidic site in quercetin, see text. (3) After addition of 8% (v/v) H2O, see text.
H
H
H
H
HO
H
O
H
O
H
O
H
H+
MeOH/H2O
OH
O
OH
O
OH
O
OH
O
NBS
Br
H
O
H
OH
-H+
O
Br
Br
Br
H+
HO
Br
O
O
H
NBS
H
H+
Br
OH
O
OH
O
OH
O
HO
OH
O
Scheme 3. Mechanism of bromination of quercetin with NBS in methanol/water, see text.
the A-ring of quercetin is activated toward electrophilic substitu-
The involvement of the quercetin anion in the bromination
mechanism represented in Scheme 3 accounts for a few details of
the reaction that otherwise would be difficult to explain. If the
reactive species was the neutral quercetin, the rapid double
bromination of the A-ring, for instance, would hardly be explained
because the monobrominated species 3 and 4 would be further
deactivated as substrates of electrophilic reactions. Actually, once
the first Br atom enters the ring the second bromination step can
be further accelerated by the increased acidity of the 7-OH in the
monobromoquercetins.30 On the other hand, addition of diluted
HCl did not inhibit the bromination reaction, although to some
extent it certainly depressed the ionization of the 7-OH. This might
tions by ionizing solvents through deprotonation of the 7-OH. This
conclusion is further supported by some additional observations we
came across during the NMR experiments. Quercetin displayed a
singular hydrogen/deuterium exchange at the positions 6 and 8
that was apparently driven by the ionization of the 7-OH. We
observed that the NMR signals of the protons 8 and 6 slowly28
disappeared when a methanol-d4/D2O solution of quercetin was
treated with diluted NaOD or DCl (the details will be reported in
a forthcoming Letter), see Scheme 4. A complete isotope exchange
took place in our conditions also because the bond enthalpy of
C-D is greater than that with the lighter isotope, C–H.29

M. C. Foti, C. Rocco / Tetrahedron Letters 55 (2014) 1602–1607
1607
H
H
H
H
DO
H
O
H
O
H
O
H
D+
MeOD/D2O
OD
O
OD
O
OD
O
OD
O
D+
H
H
D
O
O
H
H
D
OD
D
O
O
OD
H
O
-H+
D
DO
D
O
H
O
D
-H+
+D+
OD
O
OD
OD
O
Scheme 4. Mechanism of H/D exchange in the A-ring of quercetin in methanol-d4/D2O containing DCl, see text.
2. Conquer, J. A.; Maiani, G.; Azzini, E.; Raguzzini, A.; Holub, B. J. J. Nutr. 1998, 128,
593–597.
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2005, 81, 230S–242S.
Br
N
Br
N
OH
OH
O
O
5. Nijveldt, R. J.; Van Nood, E.; Van Hoorn, D. E. C.; Boelens, P. G.; Van Norren, K.;
Van Leeuwen, P. A. M. Am. J. Clin. Nutr. 2001, 74, 418–425.
6. Foti, M. C. J. Pharm. Pharmacol. 2007, 59, 1673–1685.
7. Pietta, P.-G. J. Nat. Prod. 2000, 63, 1035–1042.
Scheme 5. Canonical resonance structures of protonated NBS.
8. In the last decade, more than 2600 articles regarding quercetin can be found in
literature (data from Scopus).
9. Foti, M. C.; Daquino, C.; Dilabio, G. A.; Ingold, K. U. Org. Lett. 2011, 13, 4826–4829.
10. Sokolovà, R.; Ramesovà, S.; Degano, I.; Hromadovà, M.; Gal, M.; Zabka, J. Chem.
Commun. 2012, 3433–3435.
11. Oliveira Brett, A. M.; Ghica, M.-E. Electroanalysis 2003, 15, 1745–1750.
12. Boersma, M. G.; Vervoort, J.; Szymuslak, H.; Lemanska, K.; Tyrakowska, B.;
Cenas, N.; Segura-Aguilar, J.; Rietjens, I. M. C. M. Chem. Res. Toxicol. 2000, 13,
185–191.
13. Toteva, M. M.; Richard, J. P. J. Am. Chem. Soc. 2000, 122, 11073–11083.
14. Toteva, M. M.; Moran, M.; Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc. 2003, 125,
8814–8819.
15. Askal, H. F.; Saleh, G. A.; Backheet, E. Y. Talanta 1992, 39, 259–263.
16. Saleh, G. A.; El-Shaboury, S. R.; Mohamed, F. A.; Rageh, A. H. Spectrochim. Acta A
2009, 73, 946–954.
17. Mohamed, F. A.; Hussein, S. A.; Mohamed, H. A.; Ahmed, S. A. Bull. Pharm. Sci.,
Assiut University 2003, 26, 15–27.
18. Nagimova, A. D.; Zhusupova, G. E.; Erzhanova, M. S. Chem. Nat. Compd. 1996, 32,
695–697.
19. Kleckner, I. R.; Foster, M. P. Biochim. Biophys. Acta 2011, 1814, 942–968.
20. Daquino, C.; Foti, M. C. Tetrahedron 2006, 62, 1536–1547.
21. We attempted at purifying the precipitate containing the free phenols 5 and 2
by chromatography over silica diol using acetone/hexane as an eluent.
However, phenol 5 decomposed and we were forced to acetylate the mixture
prior to chromatographic separation.
indicate that the acidity of the 7-OH can be comparatively larger
than previously reported26 but we cannot really exclude that under
(strongly) acidic conditions bromination may occur without
involvement of the quercetin anion.
Yet, addition of HCl may also induce an increase in the reactivity
of NBS, that is, in the electrophilicity of its Br atom, through
protonation of one carbonyl group, see Scheme 5. It is known in
fact that highly deactivated aromatic compounds can indeed be
smoothly monobrominated by treatment with NBS in concentrated
H2SO4 medium.31
In conclusion, bromination of quercetin with NBS in methanol/
water occurs rapidly in the A-ring only and yields—through a
simple one-pot and environmentally-friendly synthesis—various
interesting brominated derivatives by changing the pH or the
organic solvent. We have advanced a reasonable mechanism which
is based on the prior ionization of the 7-OH and on the successive
electrophilic attack of Br at the electron-rich positions 8 and 6 of
the quercetin anion. A singular H/D exchange at the position 8
and (more slowly) at the position 6 of quercetin occurs reasonably
with a similar mechanism. All these findings fully confirm the com-
paratively high acidity of the 7-OH relative to the other OHs. Excess
of NBS over quercetin produces intensely colored and surprisingly
stable bromoquinones/bromoquinomethides. Finally, the mecha-
nisms described yield bromoderivatives that can be useful inter-
mediates for the regioselective functionalization of quercetin and
other flavonoids.
22. Timbola, A. K.; de Souza, C. D.; Giacomelli, C.; Spinelli, A. J. Braz. Chem. Soc.
2006, 17, 139–148.
23. Bondzic, A. M.; Lazarevic-Pasti, T. D.; Bondzic, B. P.; Colovic, M. B.; Jadranin, M.
B.; Vasic, V. M. New J. Chem. 2013, 37, 901–908.
24. Zhang, Y.-Z.; Paterson, Y.; Roder, H. Protein Sci. 1995, 4, 804–814.
25. See the Supporting information of Ref.
9 for more details. In brief, the
resonances at ca. 6.45 and 6.24 due to H-8 and H-6 (in 80% methanol-d4/D2O),
respectively, after addition of 0.5 equiv of NaOD shifted upfield by ca. 0.2 ppm.
This large shift is most likely due to the fact that the negative charge in the
quercetin monoanion is essentially delocalized in the A-ring, therefore
suggesting that ionization takes place at the 7-OH. Further, the C-7 signal,
upon addition of NaOD, shifted downfield toward the carbonyl (C-4) signal by
more than 5 ppm (163.68–168.74 ppm). Again, this confirms that ionization
occurs at 7-OH because the C7-Oꢂ anion has a strong C@O character in the
resonance hybrid of the anion, see Ref. 9.
Supplementary data
26. Musialik, M.; Kuzmicz, R.; Pawłowski, T. S.; Litwinienko, G. J. Org. Chem. 2009,
74, 2699–2709. and references therein.
27. Guo, G.; Lin, F. J. Hazard. Mater. 2009, 170, 645–651.
28. The rate of H-8 exchange was higher than the H-6 one. Complete H/D exchange
required, at room temperature, hours to a few day according to the quantity of
DCl or NaOD used (with the base, experiments were done under argon).
29. Jones, W. D. Acc. Chem. Res. 2003, 36, 140–146.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.
01.081.
References and notes
30. Han, J.; Tao, F.-M. J. Phys. Chem. A 2006, 110, 257–263.
31. Rajesh, K.; Somasundaram, M.; Saiganesh, R.; Balasubramanian, K. K. J. Org.
Chem. 2007, 72, 5867–5869.
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