Abundance and Reactivity of Dibenzodioxocins in Softwood
DIMITRIS S. ARGYROPOULOS,*,†,‡LUBO JURASEK,†Lı ´VIA KRIS ˇTOFOVA Ä,‡
ZHICHENG XIA,§YUJUN SUN,†,‡AND ERNEST PALUS ˇ‡
Department of Chemistry, Pulp and Paper Research Centre, McGill University, 3420 University Street,
Montreal, Quebec, Canada H3A 2A7, and Paprican, 570 Boulevard St-Jean, Pointe-Claire,
Quebec, Canada H9R 3J9
To define the abundance and comprehend the reactivity of dibenzodioxocins in lignin, model compound
studies, specific degradation experiments on milled wood lignin, and molecular modeling calculations
have been performed. Quantitative31P NMR measurements of the increase of biphenolic hydroxyl
groups formed after a series of alkaline degradations in the presence of hydrosulfide anions (kraft
conditions) showed the presence of 3.7 dibenzodioxocin rings/100 C9 units in milled wood lignin.
The DFRC degradation protocol (Derivatization Followed by Reductive Cleavage) was chosen as an
independent means to estimate their abundance. Initial experiments with a dibenzodioxocin model
[1,4]dioxocin-6-ylmethanol, showed that it is not cleaved under DFRC conditions, but rather it
isomerizes into a cyclic oxepine structure. Steric effects precluded this isomerization from occurring
when DFRC was applied to milled wood lignin. Instead, monoacetylated biphenolic moieties were
released and quantified by31P NMR, at 4.3 dibenzodioxocin rings/100 C9 units. The dibenzodioxocin
content in residual lignins isolated from kraft pulps delignified to various degrees showed that during
pulp delignification, the initial rate of dibenzodioxocin removal was considerably greater than the
cleavage rate of arylglycerol-?-aryl ether bonds. The activation energy for the degradation of
dibenzodioxocins under kraft conditions in milled wood lignin was 96 ( 9 kJ/mol, similar to that of
arylglycerol-?-aryl ether bond cleavage.
dibenzodioxocin; HMBC; HMQC; model compound; kraft pulping; lignin; NMR;31P;13C; oxepine
5,5′-Biphenyl; activation energy; derivatization followed by reductive cleavage (DFRC);
Lignin is an aromatic biopolymer that constitutes ∼30% of
the dry weight of softwoods and ∼20% of the weight of
hardwoods (1). The structure of native lignin cannot be fully
characterized because of the difficulty of isolating native lignin
from the plant cell walls. Milled wood lignin is considered to
be the most representative lignin preparation (2, 3). Chemically,
lignin is built from phenylpropanoid units linked together by
various bonds. ?-O-4-coupling of a monolignol with the growing
lignin polymer creates the most abundant structural unit in
softwood lignin, the ?-aryl ether. Because ∼50% of the
phenylpropanoid units in lignin are involved in ?-O-4-structures,
the cleavage of these bonds is essential for delignification (4).
Reactions of lignin and lignin model compounds under pulping
conditions have been studied extensively (5, 6). Phenylcoumaran
(?-5) structural units with 9-12% of total phenylpropanoid units
are also prominent in softwood lignins (7). The proportion of
phenylpropanoid units in biphenyl structures in softwoods ranges
from 19 to 26% (8-10). The percentage of free biphenolic
hydroxyl groups obtained by quantitative NMR spectroscopy
is 5-8%, which indicates that the biphenyl structures in
softwood lignins are etherified to a large extent (10-12).
In 1995, Brunow’s group discovered that the majority of the
o,o-dihydroxybiphenyl structures are etherified with phenyl-
propanoid units in lignin as eight-membered ring dibenzodioxo-
cin structures (13, 14). Dibenzodioxocins are now proposed to
be the main branching points in softwood lignin (13).
During the past decade lignin structural inquiries have been
greatly facilitated by the development of various degradative
protocols, such as hydrogenolysis (1), acidolysis (15), thio-
acidolysis (16), and DFRC (Derivatization Followed by Reduc-
tive Cleavage) (17). Nondegradative protocols, such as NMR,
have also been used. DFRC, which efficiently cleaves the R-
and ?-aryl ethers in lignins, releasing analyzable monomers for
quantification, is a powerful degradative method developed
recently by Lu and Ralph (17). The DFRC method uses different
* Corresponding author [telephone (514) 398-6178; fax (514) 398-8254;
†Department of Chemistry, Pulp and Paper Research Centre, McGill
§Department of Chemistry, McGill University.
658J. Agric. Food Chem . 2002, 50, 658−666
10.1021/jf010909g CCC: $22.00©2002 Am erican Chem ical Society
Published on Web 01/12/2002
chemistry for ether cleavage and is therefore a useful alternative
to the solvolytic methods. In a recent publication, the combina-
tion of DFRC with quantitative31P NMR was shown to have
significant potential for the determination of arylglycerol-?-
aryl ether and other linkages in lignins (18).
The presence of dibenzodioxocins in wood may have serious
implications with respect to the reactivity of lignin during wood
pulping and biodegradation and possibly during pulp bleaching.
In this respect it is crucial to have a protocol for the determi-
nation of these units in lignin and to accurately determine their
abundance. With this objective we examined the reactivity of
dibenzodioxocins in models and within lignin and have arrived
at an estimate for their abundance and propose a protocol for
MATERIALS AND METHODS
Materials. Acetyl bromide (Eastman Kodak Co.), acetic anhydride
99% (J. T. Baker), ammonium chloride 99% (BDH Inc.), glacial acetic
acid (99.7%), zinc dust (97.4%), dioxane (99.9%), methylene chloride
(99.99%), ethyl acetate (99.9%), and orthophosphoric acid (85%) were
purchased from Fisher Scientific. 3,4-Dimethoxytoluene (96%), used
as an internal standard in HPLC quantifications, 2-chloro-4,4,5,5-
tetramethyl-1,3,2-dioxophospholane (95%), used as a phosphitylation
reagent, cholesterol, used as an internal standard for31P NMR spectra
(99+%), sodium hydroxide (99.99%), and sodium sulfide nonahydrate
(99.99%) were all purchased from Aldrich. The water used in this study
was first distilled and then passed through a NANO Pure analytical
deionization system (Barnstead).
Synthesis of Dibenzodioxocin Model Compound 1. (1) Catalytic
Hydrogenation of Isoeugenol. Isoeugenol (50 g) was dissolved in 200
mL of absolute ethanol, and 0.5 g of 10 wt % Pd/C was added. The
reaction was carried out in a 1 L PARR (high-pressure) reactor at room
temperature with an initial hydrogen pressure of 3.5 atm. After 1 h,
the palladium on charcoal powder was filtered off and the filtrate
evaporated to give propylguaiacol as a homogeneous oil in nearly
(2) Dimerization of Propylguaiacol. Sodium acetate (CH3COONa;
18.0 g) dissolved in 100 mL of deionized water was placed into a 2 L
three-neck flask equipped with a magnetic stirrer. Propylguaiacol (16.8
g) was then added to the solution, and the total volume of water was
adjusted to 1 L. Potassium ferricyanide [K3Fe(CN)6; l.72 g] dissolved
in 300 mL of deionized water was then added dropwise to the reaction
mixture under vigorous stirring. Stirring was continued at room
temperature for 24 h. At the end of reaction, 200 mL of dichloromethane
was added into the reaction flask. The water layer was extracted three
times with methylene chloride (200, 100, and 100 mL). The combined
organic layers were washed with a saturated solution of sodium chloride
(NaCl), dried with sodium sulfate (Na2SO4), and filtered, and finally
the methylene chloride was evaporated under reduced pressure. The
raw reaction product was purified by column chromatography [300 g
of silica gel, 130-260 mesh, 2 L of solvent (eluent ) dichloromethane/
acetic acid 100:0.5) (yield ) 60%)]: δH(200 MHz; CDCl3) 0.93 (6H,
t, CH3), 1.62 (4H, m, CH2), 2.52 (4H, t, CH2), 3.85 (6H, s, OCH3),
6.69 (2H, s, ArH), and 6.80 (2H, s, ArH) (23).
Oxidative coupling of dehydrodipropylguaiacol and coniferyl
alcohol was performed according to the published method (14) to
produce the required dibenzodioxocin 1 in 35% yield: δH(200M Hz;
CDCl3) 0.99 (6H, m, CH3), 1.67 (4H, m, CH2), 2.64 (4H, m, CH2),
3.40-3.64 (2H, m, γ-CH2), 3.75, 3.88, 3.92 (3H, s, OCH3), 4.14 (1H,
m, ?-H), 4.55 (1H, d, J12.4, R-H), 5.69 (1H, s, ArOH), and 6.75-6.89
(7H, m, Ar-H).
Treatment of 1 under Kraft Pulping Conditions. A white liquor
solution was prepared, which contained 1.55 g of sodium sulfide (Na2S‚
9H2O) and 1.75 g of sodium hydroxide (NaOH) per 50 mL of deionized
The pulping experiments were performed in Teflon-lined 5 mL steel
bombs with white liquor. Model compound 1 (3 mg) and white liquor
(4 mL) were mixed within the bomb, and the air was replaced by
nitrogen before sealing. The bomb was then heated in an oil bath for
different lengths of time at 120, 140, and 160 °C. After cooling, the
reaction mixture was neutralized with dilute H3PO4(0.5 M) and freeze-
dried. After lyophilization, a known amount of an internal standard
was added and the product extracted with ethyl acetate. Samples for
HPLC analyses were withdrawn and filtered (Waters Sep-Pak car-
tridges). The amounts of released biphenyl 6 and unreacted dibenzo-
dioxocin 1 were determined using an internal standard [3,4-dimethoxy-
toluene (96%; Aldrich)].
Treatment of Milled Wood Lignin under Kraft Pulping Condi-
tions. The procedure was similar to that used for the model compound.
The softwood milled wood lignin (80 mg) was dissolved in 1.6 mL of
white liquor (see above). The reaction was carried out in a 5 mL
stainless steel bomb from which the air had been displaced by nitrogen.
The bomb was heated in an oil bath for various periods of time at 140
°C. At the end of the reaction period, the bomb was cooled with cold
water, and its contents were acidified to pH 2 using dilute hydrochloric
acid. The precipitate was collected by centrifugation and washed with
acidified water to remove inorganic salts. The precipitated lignin was
then freeze-dried and brought to constant weight at room temperature
under reduced pressure.
An additional part of degraded lignin was also recovered because it
was a water soluble low molecular weight lignin. The water portion
was lyophilized and extracted with high-purity unstabilized tetra-
hydrofuran (5 mL). This step was repeated three times to isolate more
lignin from the inorganic salts. All THF portions were then mixed into
a preweighed vial and allowed to evaporate at room temperature in a
well-ventilated hood (overnight). After evaporation, the residue became
a dark brown viscous oil. To obtain powdered lignin, the residue was
redissolved in a mixture of dioxane/water (60:40) and freeze-dried. Both
water-soluble and precipitated portions of lignins were dissolved in
the NMR solvent (pyridine/CDCl3) and mixed together.
Treatment of 1 under DFRC Conditions Followed by Alkaline
Hydrolysis. Acetyl bromide in acetic acid (2:8 v/v; 10 mL) was added
to dibenzodioxocin 1 (20.1 mg). The reaction mixture was stirred at
50 °C for 3 h. The solvent was then evaporated to dryness under reduced
pressure. An aqueous solution of sodium hydroxide (115 mg in 5 mL
of water) was added to the residue and stirred at 45-50 °C for 24 h.
The mixture was then neutralized with dilute hydrochloric acid to pH
∼5 and extracted with methylene chloride (three times, total volume
) 20 mL). The combined extracts were evaporated to dryness under
reduced pressure. Around 100 mg of NaOH in 3 mL of deionized water
was added to the residue and stirred for 24 h at 45-50 °C. The reaction
was neutralized by dilute HCl to pH 6, extracted three times with
dichloromethane (total volume ) 10 mL), dried over Na2SO4, and
evaporated. The residue was dried in a desiccator for 24 h.
Treatment of Milled Wood Lignin under DFRC Conditions. (Step
1) Acetyl Bromide DeriVatization. Acetyl bromide in acetic acid (1:9
v/v; 12.5 mL) was added to 50 mg of softwood milled wood lignin.
The reaction mixture was stirred at 50 °C for 3 h in a clean flask (care
for any residual traces of zinc at this point, their presence could induce
erroneous quantitative results). The solvent was then evaporated to
dryness under reduced pressure.
(Step 2) ReductiVe CleaVage. The above residue was immediately
dissolved in the acidic reduction solvent (dioxane/acetic acid/water )
5:4:1 ) v/v/v, 12.5 mL). Zinc dust (250 mg) was added, and the mixture
was stirred at room temperature for 30 min. The reaction mixture was
then quantitatively transferred to a saturated ammonium chloride
solution (50 mL) in a separatory funnel using methylene chloride (20
mL). The aqueous layer was extracted with further methylene chloride
(2 × 10 mL). The combined extracts were evaporated to dryness under
reduced pressure and placed into the desiccator for 24 h. The final
acetylation step of the standard DFRC protocol was not carried out so
that the released phenols could be determined by31P NMR.
HPLC analyses were carried out by reversed-phase HPLC using a
Supelcosil LC-18 column (250 × 4.6 mm), 5 µm. Acetonitrile/water
(pH adjusted to ∼5 with H3PO4) 3:1 was used as the mobile phase.
The detector wavelength was set at 285 nm, and the flow rate was 1
mL/min. The integrated areas from the chromatograms were compared
to determine the response factor. To calculate the amount of released
biphenyl and unreacted dibenzodioxocin from the LC chromatograms,
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Received for review July 12, 2001. Revised manuscript received
November 14, 2001. Accepted November 14, 2001.
666J. Agric. Food Chem ., Vol. 50, No. 4, 2002Argyropoulos et al.