Formation of a blue adduct between 4-tert-butyl-1,2-benzoquinone and 4- amino-N,N-diethylaniline

Article abstract of DOI:10.1016/S0040-4020(99)01036-4

The reaction of 4-tert-butyl-1,2-benzoquinone (BQ) with 4-amino-N,N- diethylaniline (ADA) produces a stable blue adduct with λ(max) 625 nm (ε 11,120 M^-1 cm^-1). This makes the reaction a sensitive analytical tool, suitable for the detection and spectrophotometric determination of this quinone even in the presence of biological compounds. The adduct was identified as 3-tert-butyl-4-(4'-diethylamino)phenylimino-6-hydroxycyclohexa- 2,5-dien-1-one. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(99)01036-4

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 659–662
Formation of a Blue Adduct between
4-tert-Butyl-1,2-benzoquinone and 4-Amino-N,N-diethylaniline
a
b
c
Luca Valgimigli,a, Gian Franco Pedulli, Salvatore Cabiddu, Enrico Sanjust and
*
Antonio Rescignoc
a
`
Dipartimento di Chimica Organica ‘A. Mangini’, Universita di Bologna, Via S. Donato 15, I-40127 Bologna, Italy
b
`
Dipartimento di Scienze Chimiche, Universita di Cagliari, 09042 Monserrato (CA), Italy
Cattedra di Chimica Biologica, Universita di Cagliari, 09042 Monserrato (CA), Italy
c
`
Received 12 August 1999; revised 1 November 1999; accepted 25 November 1999
Abstract—The reaction of 4-tert-butyl-1,2-benzoquinone (BQ) with 4-amino-N,N-diethylaniline (ADA) produces a stable blue adduct with
lmax 625 nm (e 11,120 MϪ1 cmϪ1). This makes the reaction a sensitive analytical tool, suitable for the detection and spectrophotometric
determination of this quinone even in the presence of biological compounds. The adduct was identified as 3-tert-butyl-4-(40-diethylamino)-
phenylimino-6-hydroxycyclohexa-2,5-dien-1-one. ᭧ 2000 Elsevier Science Ltd. All rights reserved.
Introduction
quinone (BQ) reacts with 4-amino-N,N-diethylaniline
(ADA) to provide a stable, intensely blue adduct. The
formation of this adduct is of particular interest because
the reaction is rapid and complete and the blue product
The analytical determination of o-quinones is of great
practical interest because of the wide occurrence of these
compounds in nature1 and their biological as well as
pharmacological importance.2–4 Nevertheless, several
limitations make their quantitative determination often
troublesome, especially because of the high reactivity of
these electron-deficient structures towards nucleophiles,5
which seriously affects their stability in any biological-
like environment. Therefore the unsubstituted 1,2-benzo-
quinone is rapidly attacked even by water and o-quinones
bearing electron-withdrawing substituents such as 4-nitro-
1,2-benzoquinone and 1,2-benzoquinone-4-carboxylic acid
cannot be isolated. Other o-quinones, like 1,2-naphtho-
quinones, are more stable in the presence of water, although
they are still very reactive compounds.
possesses
a
high molar extinction coefficient (e
11,120 MϪ1 cmϪ1) in a region (600–700 nm) free from
absorption by the vast majority of biological compounds.
So, the detection and the quantitative determination of BQ
in biological preparations become reliable and sensitive.
Indeed the reaction of BQ with ADA has been successfully
employed to reveal polyphenol oxidase activity in poly-
acrylamide gel electrophoresis, either from purified prepara-
tions11 or from crude extracts,12 as well as to develop a
spectrophotometric assay.13 Nevertheless, the actual nature
of the blue adduct was so far unknown. We report here the
result of a detailed investigation aiming to clarify the struc-
ture and some properties of the adduct between BQ and
ADA and to find analytical applications for the reaction
between aromatic amines and o-quinones.
Indeed the high reactivity of o-quinones has been exploited
in several analytical methods based on their reactions with
appropriate nucleophiles to provide coloured and relatively
stable adducts suitable for spectrophotometric determi-
nation. Among these reactants worthy of mention are
dimethylamine,6 piperidine,7 cysteine,8 3-methylbenzo-
thiazolin-2-one hydrazone9 and proline.10 These methods
offer possibilities for the determination of o-quinones,
which are themselves unstable and display only a weak
absorption around 400 nm due to an n–pء transition.
Results and Discussion
When ADA is allowed to react with BQ at room tempera-
ture, a blue product is quickly formed. This has now been
isolated and analysed with NMR, IR, mass spectrometry and
UV–Vis spectroscopy. On the basis of the results, the blue
compound can be assigned the structure 1 of 3-tert-butyl-4-
(40-diethylamino)-phenylimino-6-hydroxycyclohexa-2,5-
dien-1-one (the numbering of carbon atoms in 1 is arbitrary
and refers to the positions defined for NMR assignments)
(see Fig. 1).
We have recently reported11 that 4-tert-butyl-1,2-benzo-
Keywords: amines; condensations; quinones; substituent effects.
* Corresponding author. E-mail: valgimig@alma.unibo.it
0040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(99)01036-4

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L. Valgimigli et al. / Tetrahedron 56 (2000) 659–662
Figure 1.
The 1H and 13C NMR signals were assigned by comparison
with the spectra of authentic samples of ADA and BQ and
assignments were confirmed by computer simulation using
ACS ChemLab software. FT-IR spectra reveal the presence
of an intense and broad band centred at 3390 cmϪ1 (O–H
stretching) together with intense signals at 1659 cmϪ1 and
1631 cmϪ1 due to stretching of the CyO and CyN bonds of
the quinoneimine system.
An organic solution of the blue product can be held at room
temperature in the presence of air for several hours without
significant change in UV–Vis spectrum. Moreover, the puri-
fied solid compound is indefinitely stable in the dark at 0ЊC
and can be left at room temperature in the dark for several
days without significant degradation. Although the blue
adduct is not soluble in water, it forms a fine and very stable
suspension, which does not precipitate upon centrifugation
and does not give light scattering, therefore behaving as a
true solution as regards its use for tyrosinase photometric
quantitation. It can therefore be directly used for spectro-
photometric measurements.13 The UV–Vis spectral features
taken together with the stability of the adduct, make the
reaction of BQ with ADA very useful for the spectrophoto-
metric determination of the quinone. This is important
especially in the case of biological samples, where several
compounds can interfere with absorbance measurements in
the range 400–500 nm and in the UV region.
The mass spectrum of the purified blue adduct reveals an
intense Mϩ peak at m/z 326, which, together with the
observed fragmentation pattern, is in agreement with the
suggested structure. The UV–Vis spectrum13 is also con-
sistent with the highly conjugated structure 1, since the
value of lmax 625 nm and the molar extinction coefficient
at this wavelength (e 11,120 MϪ1 cmϪ1) are indicative of a
p–pء transition in an extended p system conjugated with
electron-releasing and -withdrawing groups (CyO and
CyN–, –NEt2 and –OH in structure 1).
In order to explore the usefulness for analytical purposes of
the reactions of o-quinones with aromatic amines, BQ has
been reacted with other aniline derivatives. Furthermore, to
determine whether the formation of a blue adduct upon
reaction with ADA is a general behaviour for quinones or
is specific to BQ, a number of quinones have been reacted
with ADA under experimental conditions similar to those
used for the reaction with BQ. Besides ADA, other aromatic
amines are found to react with o-quinones to give intensely
coloured adducts. However, the adducts obtained show
UV–Vis spectra with lmax values falling at shorter wave-
lengths, and a number of unidentified by-products are found.
The reaction products arising from BQ and aniline (3-tert-
butyl-6-hydroxy-4-phenyliminocyclohexa-2,5-dien-1-one,
3) and from BQ and 4-methoxyaniline (3-tert-butyl-6-
hydroxy-4-(40-methoxyphenyl)-iminocyclohexa-2,5-dien-
1-one, 4) were formed in poor yields (40% for 3 and 15% for
4). Therefore ADA can be considered the most interesting
reactant among the aniline derivatives tested so far. The
formation of a blue or blue-violet adduct upon reaction
with ADA is not a general behaviour of o-benzoquinones
but appears to be confined to those in which the 5-position is
available (and accessible) for nucleophilic attack. Thus, no
colour development was observed when ADA was mixed
with 3,5-di-tert-butyl-1,2-benzoquinone or with tert-butyl-
1,4-benzoquinone. In these cases no reaction takes place at
all as demonstrated by spectrophotometric observations,
showing the patterns of the unchanged quinones 30 min
after mixing. When ADA was reacted with other
o-quinones, susceptible of nucleophilic attack at the
5-position, coloured adducts absorbing in the range
When the mass spectrum is recorded on the crude reaction
product, a second, much weaker spectrum was observed,
which is clearly due to a less volatile compound of slightly
higher molecular weight. On the basis of the fragmentation
pattern and by comparison with the mass spectra recorded
for ADA and tert-butylcatechol, this compound could be
assigned the structure 2. Apparently this is an intermediate
in the formation of 1 (vide infra).
A possible reaction mechanism leading to the formation
of adduct 1 involves, as a first step, a nucleophilic
Michael-type addition of ADA on the a-b unsaturated
carbonyl system to form an adduct which quickly rear-
ranges to the catechol derivative 2. This electron-rich
structure is then easily oxidised to the final quinoneimine
product 1.
When a water suspension of the adduct 1 reacts with sodium
dithionite, ascorbic acid or zinc and dilute acetic acid, the
blue colour disappears; it is formed back quantitatively upon
standing in air or adding an excess of BQ. This indicates that
the quinoneimine adduct 1 can undergo reversible reduction
to the catechol adduct 2 and that both molecular oxygen and
excess BQ are able to oxidise 2 to 1, supporting the
suggested mechanism for the formation of the blue adduct
from ADA and BQ. As 2 is very readily and quickly
oxidised to 1, the overall stoichiometry of the adduct forma-
tion is 1:1 with respect to ADA and BQ. Only traces of 2
could be detected after ADA and BQ are mixed in a 1:1 ratio
and allowed to react in the presence of air.

L. Valgimigli et al. / Tetrahedron 56 (2000) 659–662
661
500–650 nm were observed. Nevertheless, in all cases the
reaction proceeded slowly and generated a multitude of side
products which reduced the yield to less than 10%. There-
fore, the adducts are of no analytical interest and no attempts
were made to purify and characterise them. 1,2-Naphtho-
quinones also give blue adducts with ADA and it is worth
noting that, in the case of 1,2-naphthoquinone-4-sulfonic
acid, the substituent is presumably eliminated as sulfite
ion, and the violet adduct obtained is identical to that
formed with unsubstituted 1,2-naphthoquinone (lmax
598 nm, e 7470 MϪ1 cmϪ1). Preliminary observations
suggest that in the case of 1,2-naphthoquinones the coloured
adducts arise, as expected, from a nucleophilic attack of
ADA at the 4-position.
temperature by mixing a water solution of the amine with
the quinone dissolved in dichloromethane or ethanol or
water as appropriate. Whenever a 2 phase system occurred
vigorous stirring was necessary.
1,2-Benzoquinone, 4-methyl-1,2-benzoquinone, 1,2-benzo-
quinone-4-carboxylic acid, 3,5-di-tert-butyl-1,2-benzo-
quinone and 2,3-naphthoquinone were prepared by
oxidation of the parent catechols with sodium periodate in
50 mM sodium acetate buffer (pH 5) at room temperature,
followed by extraction in dichloromethane. All the other
quinones used (1,2-naphthoquinone, 1,2-naphthoquinone-
4-sulfonic acid, 3,4,5,6-tetrachloro-1,2-benzoquinone)
were commercially available (Aldrich, Fluka).
1. nmax/cmϪ1 3390, 3013, 1659, 1631. dH (300 MHz,
CDCl3) 1.47 (1, s, 9H), 6.68 (4, s, 1H), 6.69 (7, s, 1H),
6,97 (10, d, 2H, Jˆ6.0 Hz), 6.73 (11, d, 2H, Jˆ6.0 Hz),
3.44 (13, q, 4H, Jˆ7.5 Hz), 1.22 (14, t, 6H, Jˆ7.5 Hz),
1.57 (OH, broad s, 1H). dC (75 MHz, CDCl3) 31.9 (1),
30.4 (2), 150.9 (3), 125.2 (4), 183.6 (5), 154.8 (6), 103.8
(7), 162.8 (8), 139.3 (9), 125.9 (10), 112.4 (11), 147.8 (12),
45.4 (13), 13.4 (14). m/z 326 (Mϩ, 100%); 311 (42%); 283
(37%); 269 (14%); 255 (12%); 190 (76%); 163 (14%); 149
(24%); 134 (40%); 119 (6%). HRMS calcd for C20H26N2O2
326.1994 found 326.1991. UV–Vis lmax 625 nm, e
11120 MϪ1 cmϪ1. Anal. Calcd for C20H26N2O2: C, 73.59,
H 8.03, N 8.58. Found: C 73.56, H 8.01, N 8.60.
Experimental
1H and 13C NMR spectra were recorded on a Varian Gemini
300 (or Varian Gemini 200) spectrometer using the purified
adducts dissolved in CDCl3 (Sigma). FT-IR spectra of the
purified adducts were recorded with a Perkin–Elmer FT-IR
1600 from a solution of the sample in chloroform (Aldrich),
using a KBr cell with an optical path 0.1 mm. GC–MS
analysis of the products after mixing ADA and BQ was
performed both on the crude reaction mixture and on the
purified adduct using a Hewlett Packard 5890 series II gas
chromatograph equipped with an HP 5971 mass detector.
Since the blue adduct proved to be too unstable to pass the
chromatographic column (HP5, 30 m, 0.025 mm i.d.) with
the necessary temperature programming (50–250ЊC) with-
out massive degradation, mass spectra of the reaction
product before and after purification were performed also
on a double-analyser spectrometer (Fison), EIϩ70 EV, with
direct probe-introduction of the sample. UV–Vis spectra
were recorded on a double beam Varian Cary 2300 spectro-
photometer and with a HP diode array.
2. m/z 328 (Mϩ, 100%); 313 (63%); 300 (26%); 283 (14%);
226 (35%); 190 (23%); 164 (20%); 149 (49%); 120 (15%);
119 (15%).
3. nmax/cmϪ1 3446, 3015, 1647, 1623, 1311. dH (200 MHz,
CDCl3) 1,49 (s, 9H), 6.39 (s,1H), 6.66 (s, 1H), 6.92 (d, 2H,
Jˆ8.0 Hz), 7.22 (m, 3H), 1.60 (OH, broad s, 1H). UV–Vis
(CH2Cl2) lmax 491 nm, e 3596 MϪ1 cmϪ1. Anal. Calcd for
C16H17NO2: C 75.27, H 6.71, N 5.49. Found: C 75.24, H
6.69, N 5.47.
A pure sample of the blue adduct was prepared by adding a
concentrated solution of BQ (2 mmol) in dioxane (Aldrich)
to 3 mmol of ADA sulfate (Fluka) dissolved in water. The
blue adduct was quantitatively formed (as judged from
spectrophotometric analysis) immediately after mixing at
room temperature and was extracted with dichloromethane
(Aldrich). The organic solution was washed with water,
dried over Na2SO4 and evaporated in vacuo to yield the
blue product which was purified by preparative TLC on
silica gel (Merck, 20 cm×30 cm×1 mm† by eluting with
ethyl acetate/petroleum ether 15:85. The purified product
was extracted from the plate with isopropanol which was
evaporated in vacuo at room temperature. The overall yield
of purified adduct on the basis of BQ was 87%. Since 4-tert-
butyl-1,2-benzoquinone (BQ) is not a commercial product,
it was prepared from 4-tert-butylcatechol (Fluka) by stirring
in the dark for 1 h at room temperature a concentrated diox-
ane solution with an excess of Ag2O in the presence of an
excess of dry Na2SO4. The inorganic material was then
removed by filtering and the solution was deoxygenated
by bubbling with nitrogen and evaporated in vacuo at
room temperature. No further purification was necessary.
4. nmax/cmϪ1 3438, 2961, 1645, 1630, 1321. dH (200 MHz,
CDCl3) 1.27 (s, 9H), 3.67 (s, 3H), 6.40 (s, 1H), 6.70 (s, 1H),
6.82 (m, 4H), 1.54 (OH, broad s, 1H). UV–Vis (CH2Cl2)
lmax 459 nm,
e
1974 MϪ1 cmϪ1
. Anal. Calcd for
C17H19NO3: C 71.56, H 6.71, N 4.91. Found: C 71.53, H
6.70, N 4.89.
Acknowledgements
Financial support by MURST (Rome) is gratefully acknowl-
edged. We thank Dr Luca Zuppiroli for assistance with
mass spectroscopy and Mr Alen Ianni for assistance with
chromatography.
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