The DFRC method for lignin analysis. 7. Behavior of cinnamyl end groups

Article abstract of DOI:10.1021/jf981138s

The behavior of cinnamyl end groups of lignins during the derivatization followed by reductive cleavage (DFRC) procedure has been investigated using lignin model compounds. On AcBr treatment, hydroxycinnamyl alcohols give rise mainly to 1-aryl-1,3-dibromo

Full text of DOI:10.1021/jf981138s

J. Agric. Food Chem. 1999, 47, 1981−1987
1981
Th e DF RC Meth od for Lign in An a lysis. 7. Beh a vior of Cin n a m yl
En d Gr ou p s
Fachuang Lu and J ohn Ralph*
U.S. Dairy Forage Research Center, Agricultural Research Service, U.S. Department of Agriculture, and
Department of Forestry, University of WisconsinsMadison, Madison, Wisconsin 53706
The behavior of cinnamyl end groups of lignins during the derivatization followed by reductive
cleavage (DFRC) procedure has been investigated using lignin model compounds. On AcBr treatment,
hydroxycinnamyl alcohols give rise mainly to 1-aryl-1,3-dibromopropanes from which 1-aryl-3-
bromopropanes and arylcyclopropanes are formed by zinc reduction. Arylpropene derivatives are
also significant among DFRC products of etherified cinnamyl end-group models. Major monomers
from DFRC of hydroxycinnamaldehydes are arylcyclopropyl acetates produced by reductive ring
closure of 1-acetoxy-3-aryl-1,3-dibromopropanes. Although the reactions are not as clean as the ether-
cleaving reactions that form the basis of the DFRC method, end groups produce diagnostic compounds
that provide valuable markers for studying end groups in lignins.
Keyw or d s: Acetyl bromide; lignin model compound; end groups; â-bromoether; reductive elimina-
tion; DFRC; hydroxycinnamyl alcohol; hydroxycinnamaldehyde; arylcyclopropane
INTRODUCTION
1981; Lapierre et al., 1982, 1983, 1991; Monties, 1985;
Tollier et al., 1986; Terashima and Fukushima, 1988;
Terashima and Seguchi, 1988; He and Terashima, 1990,
1991; Fukushima and Terashima, 1991; Kim et al.,
1994; Ede et al., 1996). Recently we found that a milled
tobacco lignin, like DHPs, has a high content of cin-
namyl end groups, â-5 and â-â linkages (Ralph et al.,
1998). The content of end groups in lignins is therefore
an important characteristic of lignin structure. It would
be helpful to be able to quantify lignins’ end groups for
a better understanding of lignin biosynthesis.
The DFRC (d erivatization followed by r eductive
cleavage) method is a recently developed analytical tool
for lignin characterization (Lu and Ralph, 1997a,b,
1998a-c; Peng et al., 1998, 1999; Ralph and Lu, 1998).
Through DFRC, â-aryl ether linkages in lignin are
cleaved, releasing monomers that are quantified by GC.
Most monomeric and dimeric DFRC products have been
identified. A previous study (Lu and Ralph, 1998a)
showed that monomers released from end groups were
diagnostic, but a mechanism to account for their forma-
tion was not clear. In this study several lignin models
with cinnamyl end groups were subjected to DFRC
degradation and major monomers isolated and identi-
fied. Mechanisms leading to the formation of these
diagnostic monomers are addressed.
Lignins are complex natural polymers arising from
an enzyme-mediated dehydrogenative polymerization of
phenylpropanoid precursors, primarily coniferyl and
sinapyl alcohols (Harkin, 1967; Freudenberg and Neish,
1968; Adler, 1977; Chen, 1991). Softwood guaiacyl
lignins are derived primarily from coniferyl alcohol,
whereas hardwood guaiacyl-syringyl lignins come from
a mixture of coniferyl and sinapyl alcohols. Lignification
involves free-radical coupling reactions, sometimes com-
bined with nucleophilic additions to quinone methide
intermediates, to form three-dimensional polymers.
Sarkanen (1971) reviewed the two types of polymeri-
zation processes for synthetic lignin (DHP) formation
in vitro originated by Bernd Lehman and J ohn M.
Harkin (Harkin, personal communication). The Zutropf
DHPs, formed by adding lignin precursors slowly and
continuously, were called “endwise” polymers and struc-
turally resembled isolated wood lignins more closely
than Zulauf DHPs or “bulk” polymers, formed by adding
the precursors in a single batch.
One characteristic difference between endwise and
bulk synthetic lignin polymers is that there are fewer
cinnamyl end groups in the former than in the latter,
because bulk lignification involves substantial immedi-
ate dimerization. Endwise polymerization more fre-
quently involves addition of a monomer to a growing
lignin oligomer. Because the number of cinnamyl end
groups in lignin is relatively low, lignification in the
plant cell wall is believed to be an endwise polymeri-
zation, although there is considerable evidence for
cytochemical heterogeneity in lignins (Grushnikov and
Shorygina, 1970; Terashima et al., 1979, 1986a,b; Lapi-
erre and Monties, 1981, 1991; Monties and Lapierre,
EXPERIMENTAL PROCEDURES
1
1H, 13C, and 2D H-13C correlation NMR (gradient HMQC
and HMBC) spectra were taken on a Bruker DRX-360 instru-
ment fitted with a 5-mm probe with inverse geometry (proton
coils nearest the sample) and three-axis gradients. The condi-
tions used for all samples were 2-60 mg of material in 0.4
mL of acetone-d6, with the central solvent peak as internal
reference (δH 2.04, δC 29.80). The carbon/proton designations
are based on the conventional lignin numbering system
(Figures 3 and 6).
* Address correspondence to this author at the U.S. Dairy
Forage Research Center, USDA-ARS, 1925 Linden Dr. W.,
Madison, WI 53706-1108 [telephone (608) 264-5407; fax (608)
264-5147; e-mail jralph@facstaff.wisc.edu].
GC and GC/MS conditions were as described previously (Lu
and Ralph, 1998a). Mass spectra for brominated products are
reported by specifying the bromine-derived multiplets followed
10.1021/jf981138s CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/03/1999

1982 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Lu and Ralph
by their relative intensities, e.g. 322/324/326 (4/8/4)sthe triplet
signifying an ion with two bromines.
Th e DF RC P r oced u r e. For GC/MS analysis, 5-10 mg of
substrates was used for DFRC. For preparative scale DFRC,
100-150 mg of starting materials was used.
1-(4-Acetoxy-3,5-dimethoxyphenyl)-3-bromopropane (11b): δH
2.16 (2H, m, bs), 2.74 (2H, m, R’s), 3.50 (2H, t, J ) 6.6 Hz,
γ’s), 3.80 (6H, s, OMe), 6.80 (2H, s, A2/6); δC 34.1 (γ), 35.1 (â),
35.2 (R), 56.4 (OMe’s), 106.0 (A2/6), 128.1 (A4), 140.1 (A1),
153.1 (A3/5), 168.6 (A-OAc).
4-Acetoxy-3-methoxyphenylcyclopropane (14a ): δH 0.68 (2H,
m, â/γ), 0.94 (2H, m, â/γ), 1.90 (1H, m, R), 2.19 (3H, s, A-OAc),
3.76 (3H, s, OMe), 6.66 (1H, dd, J ) 8.0, 1.8 Hz, A6), 6.801
(1H, d, J ) 1.80 Hz, A2), 6.87 (1H, d, J ) 8.0 Hz, A5); δC 9.5
(â/γ), 15.9 (R), 20.5 (A-OAc), 56.0 (OMe), 110.9 (A2), 118.0 (A6),
123.2 (A5), 138.7 (A4), 143.7 (A1), 152.1 (A3), 169.0 (A-OAc).
4-Acetoxy-3,5-dimethoxyphenylcyclopropane (14b): δH 0.70
(2H, m, â/γ), 0.93 (2H, m, â/γ), 1.89 (1H, m, R), 2.19 (3H, s,
A-OAc), 3.76 (6H, s, OMe’s), 6.4 (2H, s, A2/6); δC 9.5 (â/γ), 16.5
(R), 20.3 (A-OAc), 56.4 (OMe’s), 103.1 (A2/6), 127.7 (A4), 143.6
(A1), 153.0 (A3/5), 168.6 (A-OAc).
1-(4-Acetoxy-3-methoxyphenyl)-1-bromo-3,3-diacetoxypro-
pane (17a ): δH 1.99 (3H, s, γ-OAc), 2.00 (3H, s, γ-OAc), 2.22
(3H, s, A-OAc), 2.75 (2H, m, bs), 3.82 (3H, s, OMe), 5.27 (1H,
dd, J ) 8.2, 7.0 Hz, R), 6.85 (1H, dd, J ) 6.6, 4.7 Hz, γ), 7.04
(1H, d, J ) 8.1, 2.0 Hz, A5), 7.09 (1H, dd, J ) 8.1, 2.0 Hz, A6),
7.27 (1H, d, J ) 2.0 Hz, A2); δC 20.4 (A-OAc), 20.5 (γ-OAc),
20.6 (γ-OAc), 43.4 (â), 49.08 (R), 56.4 (OMe), 89.5 (γ), 112.6
(A2), 120.4 (A6), 123.8 (A5), 140.9 (A1), 141.1 (A4), 152.4 (A3),
168.9 (A-OAc), 169.0 (γ-OAc), 169.2 (γ-OAc).
1-(4-Acetoxy-3,5-dimethoxyphenyl)-1-bromo-3,3-diacetoxypro-
pane (17b): δH 2.04 (3H, s, γ-OAc1), 2.05 (3H, s, γ-OAc2), 2.26
(3H, s, A-OAc), 2.80 (2H, m, â’s), 3.86 (6H, s, OMe’s), 5.28 (1H,
dd, J ) 8.2, 6.8 Hz, R), 6.90 (1H, dd, J ) 6.6, 4.7 Hz, γ), 6.94
(2H, s, A2/6); δC 20.2 (A-OAc), 20.6 (γ-OAc’s), 43.4 (â), 49.6
(R), 56.7 (OMe’s), 89.6 (γ), 105.2 (A2/6), 129.8 (A4), 140.3 (A1),
153.3 (A3/5), 168.4 (A-OAc), 169.0 (γ-OAc), 169.2 (γ-OAc).
1-Acetoxy-3-(4-acetoxy-3-methoxyphenyl)-1,3-dibromopro-
pane (18a ) (two isomers): δH 2.05/2.11 (3H, s, γ-OAc’s), 2.23/
2.23 (6H, A-OAc), 3.01/3.14 (2H, m, â’s), 3.84/3.85 (OMe’s),
5.28/5.30 (1H, m, R), 6.63 (1H, dd, J ) 6.6, 6.0 Hz, γ), 6.75
(1H, dd, J ) 8.5, 4.1 Hz, γ’s), 7.11/7.11 (1H, dd, J ) 8.2, 2.0
Hz, A6’s), 7.05/7.06 (1H, d’s, J ) 8.0 Hz, A5’s), 7.30 (1H, d, J
) 2.0 Hz, A2’s); δC 20.4/20.4 (A-OAc), 20.6/20.7 (γ-OAc), 48.5/
49.1 (â), 50.5/50.9 (R), 56.4/56.4 (OMe), 74.0/75.5 (γ), 112.7/
112.8 (A2), 120.5/120.5 (A6), 123.9/124.0 (A5), 139.9/140.2 (A1),
141.2/141.2 (A4), 152.4/152.5 (A3), 168.5/168.9 (γ-OAc), 168.8/
168.9 (A-OAc).
1-Acetoxy-3-(4-acetoxy-3,5-dimethoxyphenyl)-1,3-dibromopro-
pane (18b) (two isomers): δH 2.06/2.12 (3H, s, γ-OAc’s), 2.22/
2.22 (6H, s, A-OAc’s), 3.15/3.16 (2H, m, bs), 3.82/3.82 (6H, s,
OMe’s), 5.27/5.29 (1H, m, R), 6.63 (1H, dd, J ) 6.1, 6.0 Hz, γ),
6.76 (1H, dd, J ) 8.74, 4.0 Hz, γ’s), 6.93/6.94 (2H, s, A2/6); δC
20.2/20.2 (A-OAc), 20.6/20.7 (γ-OAc), 49.0/48.5 (â), 51.4/51.0
(R), 56.6/56.6 (OMe’s), 73.9/75.5 (γ), 105.2/105.2 (A2/6), 129.9/
129.9 (A4), 139.6/139.3 (A1), 153.2/153.3 (A3/5), 168.2/168.2
(γ-OAc), 168.3/168.5 (A-OAc).
AcBr treatment conditions used were those previously
described (Lu and Ralph, 1997b).
AcBr treatment products were separated on normal-phase
preparative (2-mm thickness) TLC plates (Alltech, Deerfield,
IL) using CHCl3/EtOAc (20:1) as solvent. The major DFRC
final products 11, 14, and 23 were isolated from C18 reverse-
phase 1-mm TLC plates (Alltech) using MeOH/water, 6:4,
following normal-phase TLC (CHCl3/EtOAc, 20:1) from pre-
parative DFRC of 4-hydroxycinnamyl alcohols 1 and 4-hy-
droxycinnamaldehydes 15.
P r od u ct Ch a r a cter iza tion . Structures for compounds in
this section are in Figures 2 and 3. Mass spectra for the final
monomeric products were reported in part 2 of this series (Lu
and Ralph, 1998a).
3-Acetoxy-1-(4-acetoxy-3-methoxyphenyl)-1-bromopropane (3a ):
δH 1.97 (γ-OAc), 2.23 (A-OAc), 2.56 (2H, m, â), 3.85 (OMe),
4.14 (1H, m, γ), 5.29 (1H, dd, J ) 8.7, 6.4 Hz, R), 7.04 (1H, d,
J ) 8.0 Hz, A5), 7.08 (1H, dd, J ) 8.0, 2.0 Hz, A6), 7.25 (1H,
d, J ) 2.0 Hz, A2); δC 20.4 (γ-OAc), 20.7 (A-OAc), 39.3 (â), 52.3
(â), 56.3 (OMe), 62.8 (R), 112.7 (A2), 120.4 (A6), 123.8 (A5),
141.0 (A4), 141.4 (A1), 152.4 (A3), 168.9 (A-OAc), 170.8 (γ-OAc);
MS 344/346 (M+ 2/2), 302/304 (3/3), 243/245 (1/1), 223 (5), 205
(15), 163 (92), 150 (25), 131 (100).
1-(4-Acetoxy-3-methoxyphenyl)-1,3-dibromopropane (4a ): δH
2.23 (OAc), 2.62 (1H, m, â1), 2.83 (1H, m, â2), 3.57 (2H, m,
γ’s), 3.85 (OMe), 5.31 (1H, dd, J ) 9.0, 5.8 Hz, R), 7.05 (1H, d,
J ) 8.1 Hz, A5), 7.10 (1H, dd, J ) 8.1, 2.0 Hz, A6), 7.26 (1H,
d, J ) 2.0 Hz, A2); δC 20.4 (OAc), 31.9 (γ), 42.7 (â), 53.7 (R),
56.4 (OMe), 112.7 (A2), 120.4 (A6), 123.9 (A5), 140.7 (A1), 141.1
(A4), 152.4 (A3), 168.9 (A-OAc).
1-(4-Acetoxy-3,5-dimethoxyphenyl)-1,3-dibromopropane (4b):
δH 2.23 (OAc), 2.62 (1H, m, â1), 2.83 (1H, m, â2), 3.57 (2H, m,
γ’s), 3.83 (6H, s, OMe’s), 5.28 (1H, dd, J ) 9.0. 5.8, R), 6.90
(2H, s, A2/6); δC 20.2 (OAc), 31.9 (γ), 42.6 (â), 54.2 (R), 56.6
(OMe’s), 105.1 (A2/6), 129.8 (A4), 140.1 (A1), 153.2 (A3/5), 168.4
(A-OAc); MS 364/366/368 (M+, 1/2/1), 322/324/326 (4/8/4), 243/
245 (100/98), 164 (24), 150 (12), 149 (11), 137 (35), 129 (33),
103 (19), 91 (20), 77 (17).
4-Acetoxy-3-methoxycinnamyl bromide (5a ): δH 2.23 (OAc),
3.83 (OMe), 4.24 (1H, dd, J ) 7.8, 0.9 Hz, γ), 6.49 (1H, dt, J )
15.5, 7.8 Hz, â), 6.72 (1H, d, J ) 15.5 Hz, R), 7.01 (1H, m, A5),
7.01 (1H, m, A6), 7.20 (1H, s, A2); δC 20.4 (OAc), 34.3 (g), 56.2
(OMe), 111.2 (A2), 120.1 (A6), 123.7 (A5), 126.5 (â), 134.5 (R),
135.7 (A1), 140.9 (A4), 152.3 (A3), 168.8 (OAc); MS 284/286
(M+, 3/3), 242/244 (5/5), 163 (70), 131 (100), 103 (33).
4-Acetoxy-3,5-dimethoxycinnamyl bromide (5b): δH 2.23
(OAc), 3.82 (OMe), 4.25 (2H, dd, J ) 7.6. 0.8 Hz, γ), 6.53 (1H,
dt, J ) 15.5, 7.8 Hz, â), 6.70 (1H, d, J ) 15.5 Hz, R), 6.84 (2H,
s, A2/6); δC 20.2 (OAc), 34.3 (γ), 56.4 (OMe), 104.3 (A2/6), 126.7
(â), 129.9 (A4), 135.0 (R), 135.1 (A1), 153.3 (A3), 168.4 (OAc).
1-Acetoxy-1-(4-acetoxy-3-methoxyphenyl)-2,3-dibromopro-
pane (6a ): δH 2.12 (R-OAc), 2.23 (A-OAc), 3.80 (1H, m, γ1), 3.85
(OMe), 3.92 (1H, m, γ2), 4.80 (1H, dt, J ) 6.1, 5.9 Hz, â), 6.10
(1H, d, J ) 6.1 Hz, R), 7.08 (1H, m, A5), 7.08 (1H, m, A6), 7.25
(1H, s, A2); δC 20.5 (R-OAc), 20.8 (A-OAc), 34.7 (γ), 55.2 (â),
56.3 (OMe), 75.8 (R), 112.8 (A2), 120.7 (A6), 123.5 (A5), 136.0
(A1), 141.2 (A4), 152.2 (A3), 168.8 (A-OAc), 169.6 (R-OAc); MS
422/424/426 (M+, 2/4/2), 380/382/384 (6/12/6), 221 (20), 195 (11),
179 (32), 153 (100), 131 (12).
1-(4-Acetoxy-3-methoxyphenyl)-1,2,3-tribromopropane (7a ):
not isolated; structure from GC/MS only; MS 400/402/404/406
(M+ - CH2CdO, 4/6/6/3), 321/323/325 (36/70/37), 242/244 (6/
6), 241/243 (4/4), 163 (100), 131 (100), 103 (38).
1-(4-Acetoxy-3-methoxyphenyl)-3-bromopropane (11a ): δH
2.16 (2H, m, bs), 2.75 (2H, m, R’s), 3.49 (2H, t, J ) 6.6 Hz,
γ’s), 3.80 (3H, s, OMe), 6.80 (1H, dd, J ) 8.0, 1.8 Hz, A6), 6.95
(1H, d, J ) 8.0 Hz, A5), 6.80 (1H, d, J ) 1.80 Hz, A2); δC 34.0
(γ), 34.5 (R), 35.1 (â), 56.1 (OMe), 113.6 (A2), 121.1 (A6), 123.4
(A5), 139.2 (A4), 140.5 (A1), 152.1 (A3), 169.1 (A-OAc).
4-Acetoxy-3-methoxy-9-bromocinnamyl acetate (19a ): δH 2.07
(3H, s, γ-OAc), 2.23 (3H, A-OAc), 3.82 (3H, s, OMe), 6.33 (1H,
dd, J ) 16.1, 6.1 Hz, â), 6.88 (1H, d, J ) 16.1 Hz, R), 7.04 (1H,
d, J ) 8.2 Hz, A5), 7.10 (1H, dd, J ) 8.2, 2.1 Hz, A6), 7.25
(1H, dd, J ) 6.1, 1.0 Hz, γ), 7.27 (1H, d, J ) 2.1 Hz, A2); δC
20.4 (A-OAc), 20.7 (γ-OAc), 56.3 (OMe), 90.17 (γ), 111.6 (A2),
120.6 (A6), 123.4 (â), 123.9 (A5), 135.2 (A1), 135.2 (R), 141.5
(A4), 152.6 (A3), 168.8 (A-OAc), 169.1 (γ-OAc).
1-(4-Acetoxy-3-methoxyphenyl)-1,3,3-tribromopropane (21a ):
δH 2.23 (OAc), 3.17 (1H, m, â1), 3.40 (1H, m, â2), 3.80 (OMe),
5.26 (1H, dd, J ) 8.9, 5.5 Hz, R), 5.90 (1H, dd, J ) 8.2, 5.5 Hz,
γ), 7.07 (1H, d, J ) 8.2 Hz, A5), 7.15 (1H, dd, J ) 8.2, 2.0 Hz,
A6), 7.35 (1H, d, J ) 2.0 Hz, A2); δC 20.4 (OAc), 43.7 (γ), 52.6
(â), 54.0 (R), 56.4 (OMe), 113.0 (A2), 120.7 (A6), 124.0 (A5),
139.5 (A1), 141.4 (A4), 152.6 (A3), 168.8 (OAc).
1-(4-Acetoxy-3,5-dimethoxyphenyl)-1,3,3-tribromopropane
(21b): δH 2.22 (OAc), 3.17 (1H, m, â1), 3.42 (1H, m, â2), 3.84
(6H, s, OMe’s), 5.24 (1H, dd, J ) 9.1, 5.4 Hz, R), 5.90 (1H, dd,
J ) 8.3, 5.4 Hz, γ), 6.98 (2H, s, A-2/6); δC 20.2 (OAc), 43.8 (γ),
53.2 (â), 53.9 (R), 56.7 (OMe’s), 105.5 (A2/6), 130.2 (A4), 138.9
(A1), 153.4 (A3/5), 168.4 (OAc).
cis-1-Acetoxy-2-(4-acetoxy-3-methoxyphenyl)cyclopropane (cis-
23a ): δH 1.23 (1H, m, â1), 1.30 (1H, m, â2), 1.78 (3H, s, γ-OAc),

DFRC Products of Cinnamyl End Groups in Lignin
J. Agric. Food Chem., Vol. 47, No. 5, 1999 1983
2.20 (3H, s, A-OAc), 2.26 (1H, m, R), 3.79 (3H, s, OMe), 4.28
(1H, m, γ), 6.78 (1H, dd, J ) 8.0, 1.8 Hz, A6), 6.91 (1H, d, J )
8.0 Hz, A5), 6.92 (1H, d, J ) 1.8 Hz, A2); δC 11.4 (â), 20.5 (A-
OAc), 20.5 (γ-OAc), 22.3 (R), 53.8 (γ), 56.1 (OMe), 113.9 (A2),
121.3 (A6), 122.9 (A5), 136.6 (A1), 139.3 (A4), 151.7 (A3), 169.0
(A-OAc), 171.4 (γ-OAc).
tra ns-1-Acetoxy-2-(4-a cetoxy-3-methoxyphenyl)cyclopro-
pane (trans-23a ): δH 1.22 (1H, m, â1), 1.30 (1H, m, 2), 1.98
(3H, s, γ-OAc), 2.20 (3H, s, A-OAc), 2.20 (1H, m, R), 3.80 (3H,
s, OMe), 4.14 (1H, m, γ), 6.73 (1H, dd, J ) 8.0, 1.8 Hz, A6),
6.90 (1H, d, J ) 8.0 Hz, A5), 6.92 (1H, d, J ) 1.8 Hz, A2); δC
14.9 (â), 20.4 (A-OAc), 20.7 (γ-OAc), 23.6 (R), 56.1 (OMe), 56.5
(γ), 111.8 (A2), 119.1 (A6), 123.4 (A5), 139.3 (A4), 139.9 (A1),
152.2 (A3), 169.0 (A-OAc), 171.5 (γ-OAc).
cis-1-Acetoxy-2-(4-a cetoxy-3,5-dimethoxyphenyl)cyclopro-
pane (cis-23b): δH 1.28 (2H, m, â’s), 1.80 (3H, s, γ-OAc), 2.19
(3H, s, A-OAc), 2.25 (1H, m, R), 3.77 (6H, s, OMe’s), 4.28 (1H,
m, γ), 6.56 (2H, s, A2/6); δC 11.5 (â), 20.2 (A-OAc), 20.6 (γ-
OAc), 22.8 (R), 53.9 (γ), 56.4 (OMe’s), 106.2 (A2/6), 128.3 (A4),
136.2 (A1), 152.7 (A3/5), 168.6 (A-OAc), 171.4 (γ-OAc).
trans-1-Acetoxy-2-(4-acetoxy-3,5-dimethoxyphenyl)cyclopro-
pane (trans-23b): δH 1.27 (2H, m, â’s), 2.00 (3H, s, γ-OAc), 2.19
(3H, s, A-OAc), 2.20 (1H, m, R), 3.77 (6H, s, OMe’s), 4.17 (1H,
m, γ), 6.53 (2H, s, A2/6); δC 15.0 (â), 20.2 (A-OAc), 20.7 (γ-
OAc), 24.1 (R), 56.4 (OMe’s), 56.6 (γ), 104.1 (A2/6), 128.2 (A4),
139.5 (A1), 153.1 (A3/5), 168.6 (A-OAc), 171.5 (γ-OAc).
Syn th esis of â-Ar yl Eth er Lign in Mod els (F igu r e 5).
Etherified end-group lignin models 30 and 31 (Figure 6) were
synthesized according to published methods (Ralph and Young,
1981; Kulkarni and Sebastian, 1990; Helm and Ralph, 1992;
Lu and Ralph, 1998d).
â-Coniferaldehyde ether of guaiacyl glycol [1-(4-hydroxy-3-
methoxyphenyl)-2-[2-methoxy-4-(prop-2-enal)phenoxy]ethanol
(30a )]: δH 3.89 (3H, s, B-OMe), 3.93 (3H, s, A-OMe), 4.15 (2H,
m, Aâ), 4.55 (1H, d, J ) 4.1 Hz, AR-OH), 5.05 (1H, m, AR),
6.71 (1H, dd, J ) 15.8, 7.8 Hz, Bâ), 6.84 (1H, d, J ) 8.4 Hz,
A5), 6.97 (1H, dd, J ) 8.4, 1.8 Hz, A6), 7.09 (1H, d, J ) 8.4
Hz, B5), 7.18 (1H, d, J ) 1.8 Hz, A2), 7.27 (1H, dd, J ) 8.4,
2.1 Hz, B6), 7.38 (1H, d, J ) 2.1 Hz, B2), 7.60 (1H, d, J ) 15.8
Hz, BR), 9.68 (1H, d, J ) 7.6 Hz, Bγ); δC 56.2 (A-OMe), 56.4
(B-OMe), 72.5 (AR), 75.6 (Aâ), 111.0 (A2), 111.9 (B2), 114.3
(B5), 115.4 (A5), 119.9 (A6), 124.3 (B6), 127.6 (Bâ), 128.5 (B1),
134.0 (A1), 146.9 (A4), 148.1 (A3), 150.9 (B3), 152.4 (B4), 153.6
(BR), 193.9 (Bγ).
â-Sinapaldehyde ether of guaiacyl glycol [1-(4-hydroxy-3-
methoxyphenyl)-2-[2,6-dimethoxy-4-(prop-2-enal)phenoxy]etha-
nol (30b)]: δH 3.78 (1H, m, Aâ1), 3.82 (3H, s, A-OMe), 3.93 (6H,
s, B-OMe’s), 4.28 (1H, m, Aâ2), 4.31 (1H, d, J ) 2.4 Hz, AR-
OH), 4.84 (1H, dt, J ) 8.7, 2.6 Hz, AR), 6.76 (1H, dd, J ) 15.9,
7.6 Hz, Bâ), 6.77 (1H, d, J ) 8.1 Hz, A5), 6.84 (1H, dd, J )
8.1, 1.8 Hz, A6), 7.03 (1H, d, J ) 1.8 Hz, A2), 7.11 (2H, s, B2/
6), 7.60 (1H, d, J ) 15.9 Hz, BR), 9.67 (1H, d, J ) 7.6 Hz, Bγ);
δC 56.2 (A-OMe), 56.7 (B-OMe’s), 72.9 (AR), 80.5 (Aâ), 107.0
(B2/6), 110.8 (A2), 115.4 (A5), 119.9 (A6), 129.1 (Bâ), 131.1 (B1),
132.9 (A1), 140.5 (B4), 146.9 (A4), 148.1 (A3), 153.4 (BR), 154.4
(B3/5), 193.9 (Bγ).
â-(Coniferyl alcohol) ether of guaiacyl glycol [1-(4-hydroxy-
3-methoxyphenyl)-2-[4-(3-hydroxypropenyl)-2-methoxyphenoxy]-
ethanol (31a )]: δH 3.83 (3H, s, B-OMe), 3.84 (3H, s, A-OMe),
3.88 (1H, t, J ) 5.5 Hz, Bγ-OH), 4.04 (2H, m, Aâ), 4.21 (2H,
dt, J ) 5.5, 1.5 Hz, Bγ), 4.55 (1H, d, J ) 3.7 Hz, AR-OH), 4.98
(1H, dt, J ) 7.5, 3.7 Hz, AR), 6.27 (1H, dt, J ) 15.9, 5.5 Hz,
Bâ), 6.52 (1H, dt, J ) 15.9, 1.5 Hz, BR), 6.80 (1H, d, J ) 8.0
Hz, A5), 6.90 (2H, dd, J ) 8.1, 1.2 Hz, B6), 6.91 (1H, d, J )
8.1 Hz, B5), 6.92 (1H, dd, J ) 8.2, 1.8 Hz, A6), 7.06 (1H, d, J
) 1.2 Hz, B2), 7.12 (1H, d, J ) 1.8 Hz, A-2); δC 56.2 (A-OMe),
56.3 (B-OMe), 63.25 (Bγ), 72.53 (AR), 75.55 (Aâ), 110.9 (B2),
110.9 (A2), 115.3 (B5), 115.4 (A5), 119.8 (A6), 120.3 (B6), 129.0
(Bb), 130.1 (BR), 131.9 (B1), 134.0 (A1), 146.8 (A4), 148.1 (A3),
149.1 (B4), 150.8 (B3).
F igu r e 1. Total-ion chromatograms of DFRC products from
coniferyl and sinapyl alcohols 1a and 1b, coniferaldehyde 15a ,
and sinapaldehyde 15b.
9.2, 2.5 Hz, AR), 6.37 (1H, dt, J ) 15.8, 5.2 Hz, Bâ), 6.55 (1H,
dt, J ) 15.8, 1.3 Hz, BR), 6.77 (2H, s, B2/6), 6.78 (1H, d, J )
8.2 Hz, A5), 6.83 (1H, dd, J ) 8.2, 2.1 Hz, A6), 7.02 (1H, d, J
) 2.1 Hz, A-2); δC 56.2 (A-OMe), 56.5 (B-OMe’s), 63.2 (Bγ),
72.9 (AR), 80.5 (Aâ), 104.5 (B2/6), 110.7 (A2), 115.4 (A5), 119.8
(A6), 130.0 (BR), 130.6 (Bâ), 132.9 (A1), 134.3 (B1), 137.3 (B4),
146.8 (A4), 148.1 (A3), 154.0 (B3/5).
RESULTS AND DISCUSSION
DF R C P r od u ct s fr om Cin n a m yl Alcoh ols a n d
Cin n a m a ld eh yd es. Coniferyl alcohol 1a , sinapyl al-
cohol 1b, coniferaldehyde 15a , and sinapaldehyde 15b
were subjected to the DFRC procedure. Although such
units in lignins are completely etherified, reactions on
these phenolic models helped to elucidate some DFRC
pathways. GC/MS chromatograms of DFRC products
from these model compounds are shown in Figure 1,
structures in Figures 2 and 3. Peaks were identified by
comparison of their GC retention times with those of
authentic compounds and/or their mass spectral data
(Lu and Ralph, 1998a). The major products shown in
Figures 2 and 3 have also been isolated and their
structures authenticated by NMR (see Experimental
Procedures). The major DFRC products from coniferyl
alcohol 1a were 4-acetoxyguaiacylcyclopropane (14a )
and the guaiacylpropyl bromide 11a accompanied by
minor components 8a -10a and 12a -13a . Compound
14a coeluted with a small amount of isoeugenol acetate
â-(Sinapyl alcohol) ether of guaiacyl glycol [1-(4-hydroxy-3-
methoxyphenyl)-2-[2,6-dimethoxy-4-(3-hydroxypropenyl)phenoxy]-
ethanol (31b)]: δH 3.69 (1H, m, Aâ1), 3.81 (3H, s, B-OMe), 3.87
(6H, s, A-OMe), 3.91 (1H, d, J ) 5.7 Hz), 4.22 (3H, m, Aâ2
and Bγ’s), 4.41 (1H, d, J ) 2.1 Hz, AR-OH), 4.81 (1H, dt, J )

1984 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Lu and Ralph
Ta ble 1. P r od u cts a n d Th eir Distr ibu tion s by Weigh t
fr om AcBr Tr ea tm en t of Com p ou n d s 1 a n d 15a
wt distribution of AcBr
substrate
treatment intermediates (%)
coniferyl alcohol (1a )
88 (4a ), 9 (6a ), 3 (3a ),
trace (2a + 5a )
sinapyl alcohol (1b)
coniferaldehyde (15a )
sinapaldehyde (15b)
90 (4b), 5 (unknown),
5 (5b), trace (2b)
84 (18a ), 11 (16a +17a +19a ),
5 (21a )
87 (18b), 9 (16b+17b+20b),
4 (21b)
a
Identified products represented >90% of the total product.
F igu r e 4. Formation of arylcyclopropane compounds 14 and
23.
F igu r e 2. DFRC reactions of coniferyl alcohol 1a and sinapyl
alcohol 1b.
ated products from sinapaldehyde (compounds 12b, 24b,
and 25b) were produced in higher yield than their
counterparts (compounds 12a , 24a , and 25a ) from
coniferaldehyde. Compound 25a was barely detected in
the GC chromatogram of DFRC products from conifer-
aldehyde.
To understand the formation of DFRC monomeric
products from cinnamyl alcohols 1 and cinnamaldehydes
15, the intermediates produced during acetyl bromide
(AcBr) treatment (Figures 2 and 3) were isolated by
preparative TLC and identified by NMR (see Experi-
mental Procedures). The relative proportions by weight
among those intermediates are listed in Table 1. 4-Hy-
droxycinnamyl alcohols 1 (Figure 2) resulted primarily
in aryl-1,3-dibromopropanes 4, presumably formed
through HBr addition to the corresponding free-phenolic
precursor of cinnamyl bromide 5, formed from their
alcohols, followed by acetylation. Minor compounds
isolated were 3a and 6a . Compound 5b was isolated as
a minor component (∼5%) in the major 4b fraction (total
) 200 mg). Tiny amounts of AcBr treatment products
from sinapyl alcohol remain unknown. No syringyl
compounds 3b or 6b could be isolated or detected by
NMR. Coniferaldehyde 15a and sinapaldehyde 15b
reacted with AcBr in acetic acid in a similar way to the
alcohols, resulting in compounds 18 as major products
and 16, 17, and 21 as minor products (Figure 3). The
only difference was that 19a was detected in the minor
fraction of AcBr treatment products from coniferalde-
hyde 15a , but 20b was observed in the AcBr treatment
products from sinapaldehyde 15b.
Once AcBr treatment intermediates were known,
chemical pathways leading to the major DFRC products
became easier to understand (Figures 2 and 3). Products
11 were formed by Zn reduction of the benzyl bromides
of intermediates 4. Cyclopropanes 14 were also produced
from 4 (Figure 4) following a known reaction pathway
(Corbin et al., 1964). Products 8 and 9 may come from
Zn reduction of intermediates 5 and/or 7/6, compound
10a from 6a , and compound 12 from 3. Finally, 13a
probably resulted from partial hydrolysis of the benzyl
F igu r e 3. DFRC reactions of coniferaldehyde 15a and sinap-
aldehyde 15b.
(9a ), which could be detected in the NMR of a fraction
from the monomer mixture of the DFRC product from
coniferyl alcohol and could be separated using an
extended GC temperature program (Lu and Ralph,
1998a). The major monomers from sinapyl alcohol 1b
were the analogous compounds 8b, 14b, and 11b.
Compounds 14b and 9b were resolved in this case.
The major DFRC monomers from coniferaldehyde 15a
and sinapaldehyde 15b were cis- and trans-arylcyclo-
propyl acetates 23 (Figures 1 and 3). The minor acetyl-

DFRC Products of Cinnamyl End Groups in Lignin
J. Agric. Food Chem., Vol. 47, No. 5, 1999 1985
F igu r e 5. Scheme for syntheses of 4-O-etherified cinnamyl alcohol/cinnamaldehyde end-group model compounds 30 and 31: (i)
K2CO3, acetone; (ii) NaBH4, EtOAc; (iii) 1,4-benzoquinone, diglyme, 120 °C; (iv) NaOH, EtOH; (v) NaBH4, EtOH.
bromide on intermediate 4a followed by acetylation. The
AcBr treatment intermediates from the aldehydes 15
suggest that the major products 23 were from interme-
diates 18 through Zn reduction (Figure 4). Products 8
and 9 may result from 21, 16, and/or 19/20. Minor
products 24 could be from intermediates 17 or 18, but
how product 25b has been formed remains unknown.
Products 2 were from intermediates 16 by Zn reduction
followed by acetylation.
DF RC Mon om er s fr om Cin n a m yl Alcoh ol a n d
Cin n a m a ld eh yd e E n d Gr ou p s of â-Ar yl E t h er
Mod els 30 a n d 31. Four â-aryl ether lignin model
compounds containing etherified cinnamyl end groups,
which are better representatives of their structures in
lignins, were synthesized according to standard methods
as shown in Figure 5. Hydroxycinnamyl alcohols 1 were
synthesized by reduction of hydroxycinnamaldehydes 15
with sodium triacetoxyborohydride (Lu and Ralph,
1998d) and condensed with bromo-4-acetoxy-3-meth-
oxyacetophenone (26). The resultant ketone 27 was
reduced with sodium borohydride in ethyl acetate to the
corresponding benzyl alcohol 28. Benzoquinone oxida-
tion (Kulkarni and Sebastian, 1990) oxidized the pri-
mary alcohol group in 28 to give the aldehyde 29. Other
steps shown in Figure 5 have already been described
(Ralph and Young, 1981; Helm and Ralph, 1992). A
simple two-carbon side chain on the A moiety was used
instead of the typical three-carbon one of lignin models
because DFRC products from cinnamyl alcohol/cinna-
maldehyde end groups can be readily found and the A
side chain was not expected to significantly affect the
reaction course. In this case, the only DFRC product
from the A moieties of models 30 and 31 was the
4-acetoxy-3-methoxystyrene; the rest of the products
came from the corresponding end groups (B moiety).
F igu r e 6. Total-ion chromatograms of DFRC products from
Figure 6 shows total ion chromatograms of DFRC
products from models 30 and 31. By comparing Figure
6 with Figure 1, it was found that products from 4-O-
etherified cinnamyl end groups were similar to those
from the corresponding cinnamyl alcohols/cinnamalde-
hydes with free-phenolic groups. However, the propor-
models 30 (containing 4-O-etherified cinnamaldehydes) and
31 (containing 4-O-etherified cinnamyl alcohols): (#) 4-acetoxy-
3-methoxystyrene from the A moiety.
tions of the products were different, especially for
cinnamyl alcohol end groups. Model 31a produced more

1986 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Lu and Ralph
8a , 13a , and 9a compared to coniferyl alcohol 1a .
Similarly, model 31b gave rise to more 8b and 9b
compared to sinapyl alcohol 1b. Model 30 had patterns
of products similar to those from the corresponding
aldehydes 15, although small variations were found in
the case of 30b, from which a significant amount of one
unknown compound X and 13b were produced by the
DFRC method.
following DFRC treatment. The reactions are not as
clean as the ether-cleaving reactions that form the basis
of the DFRC method but, nevertheless, provide valuable
markers for studying end groups in lignins. Cinnama-
ldehyde end groups produce characteristic arylcyclopro-
pyl acetates, so the DFRC method could find value in
the understanding of compositional changes in mutant
and transgenic plants where aldehyde buildup is sus-
pected. Cinnamyl alcohol groups produce a mixture of
arylpropenes and 1-aryl-3-bromopropanes, along with
more diagnostic arylcyclopropanes. Coniferyl alcohol end
groups also form 1-acetoxy-1-aryl-3-bromopropanes.
Although the product mixtures are more complex, the
production of relatively diagnostic “fingerprint” products
from cinnamyl alcohol end groups also allows the DFRC
method to provide useful data on these features of
lignins.
F or m a tion a n d Id en tifica tion of th e Ar ylcyclo-
p r op a n e Com p ou n d s 14 a n d 23. The major DFRC
monomers from cinnamaldehyde end groups were di-
agnostic arylcyclopropyl acetates 23 (Figure 3), whereas
significant amounts of arylcyclopropanes 14 (Figure 2)
were produced from cinnamyl alcohol end groups (Fig-
ure 1). Each diastereoisomer of compounds 23 was
isolated by reverse-phase TLC following normal-phase
TLC from preparative-scale DFRC of cinnamaldehydes
15. Identification of 23 isomers was made by the usual
series of NMR experiments (1H, 13C, DEPT, and 2D
gradient-enhanced HMQC and HMBC). Two low-field
multiplet signals around δH 1.2-1.3 in the 1H NMR
spectra, and the corresponding δC 11.4-11.5 methylene
signals in the 13C NMR spectra, indicated the presence
of cyclopropane protons in compounds 23. Singlets
integrating for three protons at δH 1.8-2.0 indicated a
side-chain (aliphatic) acetate attached to tertiary car-
bons. In HMBC experiments, C-1 on the aromatic ring
correlated with all of the protons on the side chain, an
occurrence not encountered in typical aryl-n-propyl side
chains. All of the NMR and mass spectral data of
compounds 23 were consistent with arylcyclopropyl
acetates. Isomers were assigned on the basis of the
higher 13C shift of CR and Cγ (Breitmaier and Voelter,
1987) accompanied by a lower proton shift of Hγ due to
shielding by the aromatic ring in the trans-isomer.
Compounds 14 were isolated (in the same way as was
used for isolating compounds 23) from preparative
DFRC of cinnamyl alcohols 1. In the proton NMR
spectra of 14, two multiplet peaks at δH 0.7 and 0.9,
integrating for two protons each, indicated the presence
of two pairs of cyclopropane protons. Those protons
correlated with only one carbon peak in HMQC experi-
ments, suggesting a symmetry. Again, C-1 of the
aromatic ring correlated with all side-chain protons in
HMBC experiments. Thus, compounds 14 were identi-
fied as arylcyclopropanes.
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Received for review October 16, 1998. Revised manuscript
received February 16, 1999. Accepted February 24, 1999. We
gratefully acknowledge partial support through USDA-NRI
Competitive Grant 97-02208 (Improved Utilization of Wood
and Wood Fiber Section).
J F981138S