Y. Tobimatsu, J. Ralph et al.
solved in acetone-toluene (90 mL, 1:2, v/v) and Ag2CO3 (1.9 g,
7 mmol) was added at room temperature. After stirring at room
temperature for 14 h, the solid was filtered off, and the organic sol-
vents were evaporated under reduced pressure to give a dark solid
residue, which was purified by performing silica-gel chromatogra-
phy to give compound 2 as a colorless solid (400 mg, 1.3 mmol,
42% yield). This product was a mixture of cis- and trans-isomers of
compound 2 (cis-2/trans-2=4:96, determined by using 1H NMR).
Recrystallization from diethylether–chloroform yielded practically
pure trans-isomer.
aq. at 08C, and the augmented precipitates were collected by cen-
trifugation, washed with 0.01m HCl aq. (100 mLꢁ3), and then
lyophilized to give DHPs.
Monitoring alkaline degradation of DHPs
GPC monitoring was conducted as follows: 0.2m sodium hydroxide
(NaOH, 300 mL) aqueous solution was added to a solution (900 mL)
containing DHP (6.0 mg) in DMF at 308C. After initiating the reac-
tion, aliquots (150 mL) of the reaction mixtures were periodically
sampled, mixed with DMF (1350 mL) containing 0.1m LiBr and
0.1m acetic acid to terminate the reaction, cooled at 48C, and di-
rectly subjected to GPC analyses. For NMR monitoring, DHP
(20 mg) was dissolved in DMF (4.5 mL) and 0.2m NaOH aqueous
solution (1.5 mL) was added at 308C. After the desired reaction
time, the mixture was poured into 0.1m aq. HCl (200 mL) at 08C.
Resultant precipitates were collected by filtration through a nylon
membrane (pore size, 0.45 mm), washed with 0.01m aqueous HCl
(200 mL), and lyophilized to yield typically 16–20 mg powder. The
obtained powder was dissolved in [D6]DMSO and subjected to
NMR analyses.
1
trans-2: H NMR ([D6]acetone): d=1.16 (3H, t, J=7.58 Hz; H8’), 2.53
(2H, q, J=7.58 Hz; H7’), 3.47 (1H, m; Hg), 3.69 (1H, m; Hg), 4.00
(1H, t, J=6.02 Hz; g-OH), 4.04 (1H, m; Hb), 4.92 (1H, d, J=7.95 Hz;
Ha), 6.69 (1H, dd, J=8.15 and 1.85 Hz; H6’), 6.73 (1H, d, J=
1.80 Hz; H2’), 6.79 (1H, d, J=8.15 Hz; H5’), 6.86 (1H, d, J=8.05 Hz;
H5), 6.94 (1H, dd, J=8.05 and 1.85 Hz; H6), 7.09 (1H, d, J=1.80 Hz;
H2), 7.78 ppm (1H, s; ph-OH); 13C NMR ([D6]acetone): d=16.25
(C8), 28.67 (C7), 56.24 (OMe), 61.87 (Cg), 77.11 (Cb), 79.35 (Ca),
111.75 (C2), 115.65 (C5), 116.84 (C2’), 117.29 (C5’), 121.31 (C6’),
121.48 (C6), 129.40 (C1), 137.86 (C1’), 142.52 (C4’), 144.64 (C3’),
147.88 (C4), 148.41 ppm (C3).
cis-2: 1H NMR ([D6]acetone): d=4.45 (m; Hb), 5.24 ppm (d, J=
2.80 Hz; Ha); 13C NMR ([D6]acetone): d=76.28 (Ca), 78.29 ppm
(Cb). HR–MS (ESI) calcd. for [(MꢁH)ꢁ]: 315.1237; found: 315.1247.
Compound 3 (Figure 1) was synthesized by methylation of RA with
dimethyl sulfate: to a solution of RA (1.4 g, 4 mmol) and dimethyl
sulfate (5.7 mL, 60 mmol) in acetone (100 mL), potassium carbon-
ate (8.3 g, 60 mmol) was added, and the mixture refluxed for 12 h.
After cooling to room temperature, the solid was filtered off, and
the organic solvents were evaporated under reduced pressure to
give an oil, which was purified by performing silica-gel chromatog-
raphy to yield compound 3 as a yellowish oil (1.4 g, 3.3 mmol,
84% yield).
1H NMR ([D6]acetone): d=3.06–3.18 (2H, m; H7’), 3.69, 3.75, 3.80,
3.85, and 3.88 (15H, s; OMe), 5.25 (1H, q, J=8.40 and 4.47 Hz; H8’),
6.46 (1H, d, J=15.90 Hz; H8), 6.82 (1H, dd, J=8.15 and 1.90 Hz;
H6’), 6.86 (1H, d, J=8.15 Hz; H5’), 6.94 (1H, d, J=1.90 Hz; H2’),
6.99 (1H, d, J=8.30 Hz; H5), 7.20 (1H, dd, J=8.28 and 1.98 Hz; H2),
7.33 (1H, d, J=2.00 Hz; H2), 7.64 ppm (1H, J=15.90 Hz; H7);
13C NMR ([D6]acetone): d=37.60 (C7’), 52.32, 55.93, 55.97, 56.02,
and 56.05 (OMe), 73.77 (C8’), 110.91 (C2), 112.21 (C5), 112.54 (C5’),
114.07 (C2’), 115.37 (C8), 122.24 (C6’), 123.93 (C6), 127.92 (C1),
129.59 (C1’), 146.59 (C7), 149.33 (C4’), 150.09 (C3’), 150.56 (C3),
152.72 (C4), 166.67 (C9), 170.75 ppm (C9’). HR–MS (ESI) calcd. for
[(M+Na)+]: 453.1520; found: 453.1553.
Preparation of artificially lignified maize cell walls
Freshly prepared fully hydrated nonlignified primary cell walls
(ꢀ1.9 g dry weight) from maize cell suspensions[40] were stirred in
water (300 mL) containing 3mm CaCl2. Prior to lignification, cell
wall ferulates were dimerized through wall-bound peroxidases by
adding dilute H2O2 (0.3 mmol in 10 mL of water, ꢀ2 eq per mol of
cell wall ferulate) by using a peristaltic pump over a 30 min period
followed by stirring for an additional 30 min. Cell wall suspensions
were then artificially lignified by adding two levels of a two-com-
ponent mixture of CA and SA (each at 0.7 or 0.9 mmol) or by
adding a three-component mixture of CA and SA (each at
0.6 mmol) with RA (0.35 mmol). Lignin precursors (prepared in
10 mL dioxane and 90 mL of water) and dilute H2O2 (1.1 eq per
mol of monolignol or catechin unit prepared in 110 mL of water)
were added separately by using a peristaltic pump, initially at
20 mLhꢁ1 for 1 h, followed by 10 mLhꢁ1 for 3 h, and then complet-
ed at 5 mLhꢁ1 to mimic the proposed progression of lignin forma-
tion in plants. Treatments were replicated twice in independent ex-
periments, and nonlignified controls were stirred in a solvent mix-
ture similar to the final makeup of the lignification reaction media.
Following monolignol addition, the cell wall peroxidase activity
was visually assessed through guaiacol–H2O2 staining,[42] and the
acidity of the cell wall suspension was measured with a pH meter.
Following additions, the cell walls were stirred for an additional
12 h, stored several days at 48C, and then filtered and resuspended
four times with acetone/water [400 mL of 9:1 (v/v)] in fritted-glass
Bꢂchner funnels (ꢀ5 mm retention) to remove DHPs not bound to
cell walls. Cell walls were then dehydrated by five washes with ace-
tone (400 mL), briefly subjected to vacuum to remove excess ace-
tone, and transferred to sample jars. After setting overnight in a
hood to evaporate the acetone, cell walls were dried at 558C and
then weighed to estimate, by difference, the mass of lignin precur-
sors polymerized into cell walls. Subsamples from acetone/water
filtrates were dried in vacuo, redissolved in 1:1 (v/v) dioxane-water,
and subjected to UV spectroscopy to estimate dehydrogenation
products washed out of cell walls.
HRP-catalyzed dehydrogenative polymerization
DHPs were generated through HRP-catalyzed polymerization of CA
with RA, MC, and EC, using literature methods[39] with some minor
modifications: monomers (total 1 mmol, feed ratio listed in Table 1)
in 240 mL of acetone/sodium phosphate buffer (0.1m, pH 6.5; 1:9,
v/v) and a separate solution of hydrogen peroxide (1.2 mmol) in
240 mL of water were added by using peristaltic pump over a 20 h
period at 258C to 60 mL of buffer containing HRP (5 mg). The reac-
tion mixture was further allowed to stand at 258C for 4 h. After
polymerization with MC and EC, the precipitate was collected by
performing centrifugation (10000ꢁg, 15 min), washed with ultra-
pure water (100 mLꢁ3), and lyophilized to afford DHPs. After poly-
merization with RA, it was observed that the DHPs were partially
soluble in the final reaction mixtures, probably due to the presence
of hydrophilic carboxylic moieties in the polymers. Therefore, the
reaction mixtures were carefully acidified (pHꢀ2) using 0.1m HCl
684
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ChemSusChem 2012, 5, 676 – 686