4,4′-Dihydroxy-3,3′,5,5′-tetramethoxyazodioxybenzene: an unexpected dimer formed during hydroxylamine extractions of wheat flour

Article abstract of DOI:10.1016/j.tet.2006.06.117

Neutral hydroxylamine extracts of wheat contained a product that was colourless at pH<5 (λ_max 340 nm) and yellow at pH>9 (λ_max 400 nm). ESI-MS showed a major ion m/z 184.0 and a possible parent ion m/z 367.2 (MH⁺) suggesti

Full text of DOI:10.1016/j.tet.2006.06.117

Tetrahedron 62 (2006) 9289–9293
4,40-Dihydroxy-3,30,5,50-tetramethoxyazodioxybenzene: an
unexpected dimer formed during hydroxylamine extractions
of wheat flour
*
R. E. Asenstorfer and D. J. Mares
School of Agriculture and Wine, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA 5064, Australia
Received 31 January 2006; revised 15 May 2006; accepted 1 June 2006
Available online 4 August 2006
Abstract—Neutral hydroxylamine extracts of wheat contained a product that was colourless at pH<5 (lmax 340 nm) and yellow at pH>9
(lmax 400 nm). ESI-MS showed a major ion m/z 184.0 and a possible parent ion m/z 367.2 (MH+) suggesting that the product resulted
from the reaction of 2,6-dimethoxy-p-quinone with hydroxylamine. However, mass spectral and other spectroscopic data indicated that
the compound was neither of the 2,6-dimethoxy-p-quinone oximes. A product with identical absorbance, mass spectrum, electrophoretic
mobility and HPLC retention time as the pigment from hydroxylamine extracts of flour was observed amongst the reaction products of
hydroxylamine and 1,4-dihydroxy-2,6-dimethoxybenzene. The structure of this product was identified by NMR, 2D NMR and IR as 4,40-di-
hydroxy-3,30,5,50-tetramethoxyazodioxybenzene.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
p-quinone can be isolated from wheat germ fermented
with baker’s yeast.4,5 This compound forms two oxime
isomers, 2,6-dimethoxy-1,4-benzoquinone-4-oxime (1) and
3,5-dimethoxy-1,4-benzoquinone-4-oxime (2). These iso-
mers were synthesised and their properties compared with
those of the flour product.
Yellow alkaline noodles (YAN) made from wheat flour, alka-
line salts such as sodium and potassium carbonate and water,
are an important part of the diet in many countries of eastern
and south-eastern Asia.1 A preliminary study by Mares
et al.2 indicated that the colour of alkaline noodles was
due partly to xanthophylls and partly to compounds that
could be extracted with aqueous solvents. These latter com-
pounds, in contrast to the xanthophylls, were essentially
colourless at neutral or acid pH, but turned yellow at higher
pH. An efficient method for the quantification of these water-
soluble pigments using hydroxylamine extraction has been
recently reported.3 Together with flavone-C-glycosides,
another unknown pigment was identified as a possible con-
tributor to the colour of YAN.3 This pigment was relatively
unstable, colourless at pH<5 (lmax 340 nm) and yellow at
pH>9 (lmax 400 nm). Electrospray mass spectrum yielded
a major ion m/z 184.0 but with a possible parent ion m/z
367.2 (MH+).
O
O
1
1
H3CO
OCH3
6
6
5
2
3
2
3
5
H3CO
OCH3
4
4
N
N
HO
1
2
HO
2. Results and discussion
The two oxime isomers of 2,6-dimethoxy-p-quinone (1, 2)
were synthesised and shown to have similar mass spectra
with major ions (1) m/z (relative intensity) 367.0 (2M+H+,
0.10), 184.0 (M+H+, 0.25), 170.2 (0.10), 167.2 (0.11),
166.0 (0.68), 155.2 (0.19), 152.0 (base peak), 151.0 (0.81),
140.2 (0.25), 112.0 (0.67), 111.2 (0.26) and (2) m/z (relative
intensity) 367.0 (2M+H+, 0.07), 184.0 (M+H+, 0.20), 170.0
(0.08), 167.2 (0.36), 166.2 (0.80), 152.0 (base peak), 151.0
(0.70), 140.0 (0.09), 112.0 (0.17), 111.0 (0.26). The mass
spectra were more complex than expected and resulted from
the formation of cluster ions during the ionisation process. In
contrast, a relatively simple mass spectrum was obtained for
the product obtained from flour, which showed major ions at
If the ion-mass of 184 was an MH+ ion, it could arise from
a low molecular weight oxime formed by condensation of
hydroxylamine with either an aldehyde or ketone. One
possible carbonyl candidate, which forms an oxime with a
mass of 183, was 2,6-dimethoxy-p-quinone. 2,6-Dimethoxy-
Keywords: Wheat flour; Hydroxylamine; Hydroquinone; Oxime dimer.
* Corresponding author. Tel.: +61 8 8303 7480; fax: +61 8 8303 7109;
e-mail: robert.asenstorfer@adelaide.edu.au
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.06.117

9290
R. E. Asenstorfer, D. J. Mares / Tetrahedron 62 (2006) 9289–9293
m/z (relative intensity) 367.2 (M+H+, 0.25), 184.0 (base
peak), 156.1 (0.37), 154.0 (0.47), 139.1 (0.26), 124.1 (0.08).
These differences in fragmentation patterns, as well as dif-
ferences in absorbance spectra and HPLC retention times,
indicated that the structure of the flour product was neither
(1) nor (2). The considerably larger intensity of the mass at
m/z 367 and simpler fragmentation pattern of the flour prod-
uct compared with the oxime isomers suggested this product
was a dimer of 2,6-dimethoxy-p-quinone mono-oxime.
dimer (3) and lack of monomer–dimer equilibrium are con-
sistent with the hydroxyl groups acting as a strong electron
withdrawing substituent.6,19
The aromatic protons of the dimer show a substantially
higher d (6.51 and 6.80 ppm) when compared with 2,6-di-
methoxy-1,4-benzoquinone-4-oxime (1) (5.61 ppm) and 3,5-
dimethoxy-1,4-benzoquinone-4-oxime (2) (doublet, 5.60
and 5.63 ppm) but lower ds than literature values for either
unsubstituted or C-4 substituted cis-dimers (7.11–7.4 ppm)
and trans-dimers (7.69–7.89 ppm) of nitrosobenzenes.7
There is a significant difference (Dd 0.29 ppm) between
the aromatic protons of the dimer indicating the effect of
shielding/deshielding. In contrast, no difference was re-
corded in the shifts for the 2 and 6 protons for the aromatic
protons in dichloromethane and chloroform of either cis- or
trans-nitroso dimers.7
1-Hydroxy-2,6-dimethoxy-nitrosobenzene is a tautomeric
analogue of 2,6-dimethoxy-p-quinone oxime. Many nitroso-
benzenes dimerise under increasing concentration,6 low
temperature7 or in the presence of organic solvents8 to
give azodioxybenzenes. Nitrosobenzene trans-dimers have
a diagnostic, very strong IR band at 1253–1299 cmꢀ1 due
to the aromatic nN+–Oꢀ frequency,6,9 which is sensitive to
the molecular environment.10,11 Aromatic cis-dimers give
two active NO stretching bands at 1389–1397 cmꢀ1 and
1409 cmꢀ1. The synthetic dimeric species had a strong
band at 1316 cmꢀ1, which is consistent with the trans-
form of a nitroso dimer (Table 1). Another major band asso-
ciated with the dimer was at 952 cmꢀ1. This band12 has been
attributed to nN+–Oꢀ in compounds with aliphatic nN+–Oꢀ.
None of the dimers described by Luttke9 contained this band
and is quite specific for the compound under investigation.
It can be determined whether an isomer is either cis or trans
in solution because the shielding/deshielding of aromatic
protons in cis-N2O2C6H5ꢀnXn is virtually negligible,
whereas the shielding in the trans-N2O2C6H5ꢀnXn is sub-
stantial.7,13 Moreover, the aromatic proton signals for the
cis-dimer should be shifted to the right compared to both
the monomer and the trans-dimer. While shielding/deshield-
ing was observed for the dimer, suggesting a trans-isomer,
the aromatic proton signals were observed to be shifted to
the left compared to the monomer, but shifted to the right
compared to either cis- or trans-4-substituted nitroso-
benzene dimers.7 The higher d values of the aromatic pro-
tons of our dimer were consistent with the formation of
the azodioxybenzene compound (3). The hydroxyl group
allows the contribution of the quinonoid canonical state and
thus the aromatic protons were observed at lower d values
than other 4-substituted nitrosobenzenes. The double bond
nature of the C–N bond and the possibility of formation of
the tautomer (4) suggest that the energy barrier between
cis and trans may not be great and the dimer may exist as
both cis and trans in solution.
The IR OH stretching frequency (3300 cmꢀ1) of the dimer is
strong while the band for both oxime monomers is either
weak or missing. A strong OH stretching band indicates
that the nitroso tautomer predominates over the oxime tauto-
mer. Both oxime monomers show a medium band at approx-
imately 1345 cmꢀ1 due to nN]O stretching,6 which is
absent in the dimer.
In organic solvents, such as chloroform and dichloro-
methane, C-nitrosobenzene compounds exist in monomer–
dimer equilibria, with the cis-dimers being preferred over
the trans-dimers, irrespective of the nature of the solid-state
dimer structure.8 However, the dimer was relatively unstable
in chloroform and dichloromethane, but was stable in DMSO
and water. No monomer–dimer equilibrium was observed
in DMSO during the NMR experiments. The stability of
OH
OH
4
H3CO
3
OCH3
O
H3CO
OCH3
O
5
6
2
1
N
N
N
N
Table 1. Major peaks identified in the infrared spectra of 2,6-dimethoxy-
1,4-benzoquinone-4-oxime (1), 3,5-dimethoxy-1,4-benzoquinone-4-oxime
(2) and the dimer, 4,40-dihydroxy-3,30,5,50-tetramethoxyazodioxybenzene
(3)
O
HO
1'
2'
3'
6'
5'
H3CO
OCH3
H3CO
OCH3
4'
OH
O
3
4
1
2
3
n
—
3300w
1630vs
1575vs
1454s
1348m
—
3268vs
1644vs
1581vs
1455m
—
OH
C]O
C]N
OH bending
N]O stretch
Aryl N+–Oꢀ
The 13C NMR C1 shift of the dimer (d 148.0) was in the range
characterised by aromatic azodioxy dimers (d 139–146) and
is substantially less than the nitroso monomers (d 162–
176).14 However, the C1 shifts of the two oximes (d 137–
138) are also substantially lower than the nitroso monomers
and similar to the dimer. The exceptionally strong p-electron
acceptor nature of the N]O substituent produces astrong de-
shielding effect causing C1 shifts to higher d values, while the
N2O2 and N–OH substituents have only a moderate effect.
1626vs
1571vs
1452s
1344m
—
1316vs
C-O-CH3
1246s
1249s
1249s
1113vs
1051s
1122vs
1051vs
1125vs
1052vs
Aryl C–N stretch
C-O-CH3
The solvent used for NMR investigation is important. 13C
NMR data obtained in CDCl3 or CD2Cl2 for nitrosobenzene
monomers for the C3 and C5 and C2 and C6 carbons are
—
860m
702s
—
858m
704s
952vs
852s
702m
N+–Oꢀ
N–O bending

R. E. Asenstorfer, D. J. Mares / Tetrahedron 62 (2006) 9289–9293
9291
identical.14–16 In DMSO, however, nitrosophenol monomers
OH
N
OCH3
show differences between the C3 and C5, and the C2 and C6
carbons.17 The position of the nitroso-oxime group in 3,5-di-
methoxy-1,4-benzoquinone-4-oxime provides a larger dif-
ference between the 13C shifts of quaternary carbons (C3
and C5) than the aromatic CH carbons (C2 and C6) suggest-
ing the rotation around the C–N bond is restricted and
syn–anti isomerism is slow.18 However, 2,6-dimethoxy-
1,4-benzoquinone-4-oxime has a broad peak at 102.4 ppm
for the aromatic CH carbons (C3 and C5) suggesting the
syn–anti isomerism is rapid and splitting is minimised.
H3CO
H
H
O
N
O
H
H
H3CO
OCH3
OH
Figure 1. Correlations observed in the gHMBC spectrum of 4,40-dihydroxy-
3,30,5,50-tetramethoxyazodioxybenzene (3).
Similar to the nitrosobenzene monomers, 13C NMR studies
of azodioxybenzenes14–16 in CDCl3 and CD2Cl2 also
show no difference in the 13C chemical shifts between either
the C2 and C6 or the C3 and C5 carbons. However, in
Additional evidence for the structure of the dimer arises
from the absorbance spectral data. The molar extinction co-
efficients of the unionised oximes and the dimer are identi-
cal, while the p–p* UV band of the dimer shows a
bathochromic shift of 40 nm compared with the oxime
monomers due to a lengthening of the chromophore. In com-
parison, the p-levels of nitrosomesitylene (1,3,5-trimethyl-
2-nitrosobenzene) are not changed by dimerisation6 and
there is no effect on either lmax or extinction coefficient of
the UV bands. The C–N bond of nitrosomesitylene has sin-
gle bond character, and the nitroso group is not co-planar
with the benzene moiety and is well insulated from the benz-
ene ring in both monomer and dimer. Unlike the nitrosome-
sitylene dimer, the N+–Oꢀ group in the dimer is contiguous
with the benzene moiety and the C–N bond shows some
double bond character.
13
solid-state C NMR, trans-4,40-dichloro-azodioxybenzene
(4-ClC6H4NO)2 showed a distinction between the C2 and
C6 signals (Dd¼4.1).8 This was thought to arise from the
locked nature of the phenyl rings in the solid state and the
chemical shift represents the shielding anisotropy of the azo-
dioxy group. Similarly, the dimer (3/4) exhibited large dif-
ferences in the 13C shifts between the C3 and C5 carbons
as well as the C2 and C6 carbons when measured in
DMSO. The larger difference in shifts for the aromatic CH
carbons (C2/C20 and C6/C60) compared with the quaternary
carbons (C3/C30 and C5/C50) suggests that the azodioxy
moiety is attached proximal to the aromatic CH carbons.
gHMQC assignments together with differences in shifts
due to shielding/deshielding magnetic anisotropy allowed
the assignment of all carbons (Table 2; Fig. 1).
As most azodioxybenzenes are formed from their nitroso
monomers and in the absence of strong intramolecular
hydrogen bonding, 4-hydroxy species occur principally as
oximes20 and thus dimerisation is unlikely,9 another
mechanism is required for the formation of dimer (3). A
related compound, 4,40-dihydroxy-3,30-dimethyldiphenyl-
N,N0-dihydroxyhydrazine, detected by mass spectrometry
in photographic effluent,20 can be synthesised by reacting
2-methyl-p-benzoquinonewith hydroxylamine in a reductive
solution containing sulfite and carbonate. Therefore, a possi-
ble reaction mechanism for the synthesis of dimer (3) is
suggested (Scheme 1). A partial oxidation of 1,4-di-
hydroxy-2,6-dimethoxybenzene to create 2,6-dimethoxysemi-
quinone is necessary prior to reaction with hydroxylamine.
Table 2. Proton, carbon and gHMBC correlations of NMR of the dimer,
4,40-dihydroxy-3,30,5,50-tetramethoxyazodioxybenzene (3)
Carbon number
13C d
1H d
gHMBC
1
2
3
4
148.0
94.5
—
H2a (2b), H6a (2)
H6 (3), methyla (4)
H6 (4), methyl (3)
H2 (3), H6 (3)
H2 (4), methyl (3)
H2 (3), methyla (4)
—
6.5 (d)
—
—
—
6.8 (d)
3.71, 3.73
151.9
185.2
153.6
108.6
55.5, 55.6
5
6
Methyl
a
Weak association.
Number in brackets refers to 2-, 3-, or 4-bond correlations.
b
OH
OH
OH
OH
OH
H3CO
OCH3 H3CO
-e-, -H+
OCH3 H3CO
+ NH2OH
OCH3 H3CO
-H2O
OCH3
H3CO
OCH3
NH
OH
HO
OH
O
N
N
OH
OH
semiquinone radical
4-(hydroxyamino)-
2,6-dimethoxy-
phenol radical
OH
N
OH
N
OH
H3CO
OCH3
OH
H3CO
OCH3
OH
H3CO
OCH3
O
-2e-,-2H+
N
N
N
N
HO
HO
O
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
OH
OH
OH
Scheme 1. Suggested mechanism for the synthesis of 4,40-dihydroxy-3,30,5,50-tetramethoxyazodioxybenzene (3).

9292
R. E. Asenstorfer, D. J. Mares / Tetrahedron 62 (2006) 9289–9293
In wheat, however, it is postulated that, 1,4-dihydroxy-2,6-
dimethoxybenzene occurs as a glycoside even though this
glycoside has never been isolated.21,22 Is possible that during
the deglycosylation process, 2,6-dimethoxysemiquinone
radical is released, which then reacts with hydroxylamine.
3.4. HVPE
The electrophoretogram, consisting of chromatography pa-
per (Chr. 1; Whatman, England) soaked in the appropriate
buffer was placed over a glass rod to minimise surface con-
tact between two wells containing buffer of a gel electropho-
resis unit (Model 715; Betheseda Research Laboratories,
Gaithersburg, MD, USA). A voltage gradient of 7.5 V/cm
was used. The buffers employed were borate (0.05 mol/L;
pH 10.0) and citrate (0.05 mol/L; pH 7.0) and formic/acetic
(0.75/1.04 mol/L; pH 1.76).
3. Experimental methods
3.1. LC–mass spectrometry
The sample (5 mL) was loaded onto an HPLC column (Spher-
isorb S5 ODS2, 250ꢁ1 mm, Waters Corporation). The sepa-
ration was performed with solvent A (5% formic acid) and
solvent B (5% formic acid, 80% acetonitrile) by using a gra-
dient system: 10–35% of solvent B in 35 min, kept constant at
35% for 5 min and then 35–95% solvent B in a further
20 min, at a flow rate of 25 mL/min. The HPLC column
was connected to a UV–vis detector (HP1100, Hewlett–
Packard) monitoring at 280 and 340 nm, followed by a
mass spectrometer with an ion spray ion source (API-300,
PE Sciex, Thornhill, Ontario, Canada). The mass spectrome-
ter was operated in positive ion mode and was scanned from
m/z 250 to 1000 in 1.88 s. Ion spray and orifice potentials
were set at 5.5 kVand 30 V, respectively. Curtain and nebu-
liser gases were nitrogen and air, respectively. All mass spec-
tral data were processed using Bio-Multiview software
(version 1.2ß3, PE Sciex).
3.5. Extraction of the flour product
For direct comparison, flour (200 g) was extracted with 1 L
0.1 M hydroxylamine hydrochloride for 2 h. This was
filtered and 100 mL of the filtrate was extracted with
3ꢁ100 mL dichloromethane. The dichloromethane was
evaporated and the residue dissolved in DMSO for MS-anal-
ysis. Found: ES-MS, m/z (relative intensity) 367.2 (M+H+,
0.21), 184.1 (base peak), 156.1 (0.42), 154.0 (0.26), 139.1
(0.41) and 124.0 (0.15); HPLC retention time was
21.4 min; absorbances (aq) lmax 340 nm at pH 2.5 and lmax
400 nm at pH 10.
3.5.1. Synthesis of 2,6-dimethoxy-1,4-benzoquinone-4-
oxime (1). The title compound 1 (2,6-dimethoxy-4-oxi-
mino-2,5-cyclohexadienone-1) was synthesised from 2,6-di-
methoxy-1,4-benzoquinone and hydroxylamine according
to Bolker and Kung.23 Found: pale yellow plates; mp 214–
215 ꢂC dec (lit23 mp 218.8 ꢂC dec); ES-MS, m/z (relative in-
tensity) 367.0 (2M+H+, 0.07), 184.0 (M+H+, 0.20), 170.0
(0.08), 167.2 (0.36), 166.2 (0.80), 152.0 (base peak), 151.0
(0.70), 140.0 (0.09), 112.0 (0.17), 111.0 (0.26); HPLC reten-
tion time was 19.1 min; absorbances (aq) lmax 300 nm (3¼
16,000), 395 nm (3¼1200) at pH 2.5 and lmax 353 nm
(3¼25,300) at pH 10; dH (600 MHz DMSO-d6) 3.71 (6H,
s, OCH3), 5.61 (2H, s, H2 and H6); dC (150.9 MHz
DMSO-d6) 56.1 (OCH3), 102.4 br (C3 or C5), 137.7 (C4),
185.6 (C1), quaternary carbons (C2 and C6) were not ob-
served; nmax (KCl) 3233w, 3176w, 3064 (br), 2943w, 2750s
(br), 2644w, 2366w, 2247w, 2174w, 2100w, 1844w, 1744w,
1626vs (C]O), 1571vs (C]N), 1452s (C–O stretch),
1430m, 1403m, 1344w, 1321w, 1246s (C–O stretch),
1228s, 1192m, 1122vs (aryl C–N stretch), 1051vs (N–OH),
1019w, 986w, 930m, 908m, 860s, 810m, 795m, 702s.
3.2. IR, NMR and melting points
Infrared spectra were collected using a Perkin–Elmer (Shel-
ton, CT, USA) Spectrum 1 Fourier Transform Infrared
(FTIR) instrument equipped with a diffuse reflectance sam-
pling accessory. The data were exported to GRAMS/AI
(Thermoelectron Corporation, Woburn, MA, USA) for pro-
cessing. NMR spectra were acquired on a Varian Inova-600
NMR spectrometer, at an 1H frequency of 600 MHz and 13
C
frequency of 150 MHz. All NMR experiments were ac-
quired at 25 ꢂC. All spectra were processed on a Sun Micro-
systems Ultra Sparc 1/170 workstation using VNMR
software (version 6.1A). Melting points were obtained on
a hot stage microscope (C. Reichert Optische Werke A.G.,
Vienna, Austria).
3.3. HPLC
3.5.2. Synthesis of 3,5-dimethoxy-1,4-benzoquinone-4-
oxime (2). The title compound 2 was prepared by nitrosode-
methylation of 1,3,5-trimethoxybenzene (Sigma–Aldrich)
according to Shpinel et al.24 Found: pale yellow needles;
mp 166–167 ꢂC commenced sublimation, 216–217 ꢂC dec
(lit17 mp 223–224 ꢂC); ES-MS, m/z (relative intensity)
367.0 (2M+H+, 0.10), 184.0 (M+H+, 0.25), 170.2 (0.10),
167.2 (0.11), 166.0 (0.68), 155.2 (0.19), 152.0 (base peak),
151.0 (0.81), 140.2 (0.25), 112.0 (0.67), 111.2 (0.26);
HPLC retention time was 25.4 min; lmax (aq) 298 nm (3¼
18,000), 395 nm (3¼1400) at pH 2.5, 353 nm (3¼29,500)
at pH 10.0; dH (600 MHz DMSO-d6) 3.72 (3H, s, OCH3),
3.74 (3H, s, OCH3), 5.60 (1H, s, H2), 5.63 (1H, s, H6); dC
(150.9 MHz DMSO-d6) 56.1 (OCH3), 56.2 (OCH3), 102.1
(C2 or C6), 103.3 (C2 or C6), 137.5 (C4), 157.6 (C3 or
C5), 161.1 (C3 or C5), 185.6 (C1), in general agreement
Separation of compounds was performed using a Hewlett–
Packard HPLC 1100 instrument using a 250ꢁ4 mm analy-
tical column (Merck, Darmstadt, Germany) packed with
spherical LiChrospher 100 RP-18 (5 mm), fitted with
a 4ꢁ4 mm guard column using the same packing material.
Separation was carried out with solvent A (1% formic
acid) and solvent B (1% formic acid, 4% acetonitrile, 95%
methanol) by a gradient system elution program: 0–3 min,
isocratic 10% B; 3–8 min, gradient 10–24% B; 8–11 min,
isocratic 24% B; 11–18 min, gradient 24–34% B; 18–
28 min, gradient 34–44% B; 28–35 min, gradient 44–65%
B; 35–40 min, gradient 65–95% B; 40–55 min, isocratic
95% B; 55–60 min.
A flow rate of 0.65 mL/min,
detection at 340 and 280 nm and temperature set at 30 ꢂC
were used.

R. E. Asenstorfer, D. J. Mares / Tetrahedron 62 (2006) 9289–9293
9293
with Maleski;17 nmax (KCl) 3236w, 3182w, 3062, 2951w,
References and notes
2811w, 2800s (br), 2650w, 2249w, 2102w, 1851w,
1749w, 1630vs (C]O), 1575vs (C]N), 1465w, 1454vs
(C–O stretch), 1442, 1431, 1409s, 1348w, 1249vs (C–O
stretch), 1230s, 1194, 1125vs (aryl C–N stretch), 1066w,
1052vs (N–OH), 1019w, 988w, 934, 911, 858s, 809, 796,
704s.
1. Hou, G.; Kruk, M. AIB Technical Bulletin 1998, 20, 1–10.
2. Mares, D. J.; Wang, Y.; Cassidy, C. A., Cereals 97. Proceedings
of the 47th Cereal Chemistry Conference; RACI: Melbourne,
Australia, 1997; pp 114–117.
3. Asenstorfer, R. E.; Wang, Y.; Mares, D. J. J. Cereal Sci. 2006,
43, 108–119.
3.5.3. Reaction of 1,4-dihydroxy-2,6-dimethoxybenzene
with hydroxylamine (synthesis of 4,40-dihydroxy-
3,30,5,50-tetramethoxyazodioxybenzene 3). Approxi-
mately 10 mg of 1,4-dihydroxy-2,6-dimethoxybenzene and
20 mg hydroxylamine hydrochloride were added to 2 mL
of water and adjusted to pH 7. This mixture was allowed
to react for 16 h at 45 ꢂC. The solution was extracted with
dichloromethane. KCl was added and the dichloromethane
was carefully evaporated under reduced pressure. Found:
pale brown plates; mp 110–111 ꢂC commenced sublimation
(yellow needles), 183–184 ꢂC dec; ES-MS, m/z (relative in-
tensity): 367.2 (M+H+, 0.25), 184.0 (base peak), 156.1
(0.37), 154.0 (0.47), 139.1 (0.26), 124.1 (0.08); lmax (aq)
340 nm (3¼16,000) at pH 2.5, 400 nm (3¼17,000) at pH
10.0 (note this was estimated using HPLC and NMR data
by comparing the concentration and absorbance of 2,6-di-
methoxy-1,4-benzoquinone-4-oxime with the synthetic
product); HPLC retention time was 21.4 min; dH (600 MHz
DMSO-d6) 3.71 (6H, s, OCH3), 3.74 (6H, s, OCH3), 6.51
(2H, d, J 2.1 Hz, H2 and H20), 6.80 (2H, d, J 2.1 Hz, H6
and H60); dC (150.9 MHz DMSO-d6) 55.5 (OCH3), 55.6
(OCH3), 94.5 (C2, C20), 108.6 (C6, C60), 148.0 (C1, C10),
151.9 (C3, C30), 153.6 (C5, C50), 175.0 (C4, C40); nmax
(KCl) 3268vs (br, OH), 3092, 2965w, 2942, 2837, 2650w,
1644vs (C]O), 1581vs (C]N), 1455m (C–O stretch),
1417w, 1407, 1316vs (N+–Oꢀ), 1249vs (C–O stretch),
1219s, 1192w, 1181w, 1113vs (aryl C–N stretch), 1051,
1014, 952s (N+–Oꢀ), 907, 852s, 797, 733, 702.
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Financial support from the Grains Research & Development
Corporation and the Value Added Wheat CRC is gratefully
acknowledged. We would also like to acknowledge Yoji
Hayasaka, The Australian Wine Research Institute, for
mass spectra and Philip Clements, The University of Ade-
laide, for NMR analyses, and Dr. Max Tate, the University
of Adelaide, for helpful comments.
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