Detection and determination of p-coumaroylated units in lignins

Article abstract of DOI:10.1021/jf981140j

The derivatization followed by reductive cleavage (DFRC) method cleaves α- and β-ethers in lignins but leaves lignin γ-esters intact. When applied to grasses, which contain p-coumarate esters on their lignins, esterified monolignol derivatives are release

Full text of DOI:10.1021/jf981140j

1988
J. Agric. Food Chem. 1999, 47, 1988−1992
Detection a n d Deter m in a tion of p-Cou m a r oyla ted Un its in Lign in 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 d erivatization followed by r eductive cleavage (DFRC) method cleaves R- and â-ethers in lignins
but leaves lignin γ-esters intact. When applied to grasses, which contain p-coumarate esters on
their lignins, esterified monolignol derivatives are released. Saturation of the p-coumarate double
bond occurs during DFRC, so the released products are 4-acetoxycinnamyl 4-acetoxyphenylpropi-
onates. Synthesis of the esters allowed determination of response factors for the released products.
Maize and bamboo lignins released 221 and 38 µmol/g of p-coumarate-derived esters. The sinapyl
ester was much more abundant than the coniferyl one. The bamboo and maize lignin S/G ratios in
the conjugates were 12 and 38 times greater than those of the normal monomers released by DFRC,
evidence of a strong selectivity for acylation of syringyl units. Of three possible biochemical
mechanisms for incorporating p-coumarates into lignin, evidence is mounting that the process
involves incorporation of preacylated monolignols into the normal lignification process.
Keyw or d s: Acetyl bromide; lignin; grass; Gramineae; maize; bamboo; bromegrass; p-coumarate;
phenolic acid; â-aryl ether; cleavage; quantitative analysis; gas chromatography; reductive elimination;
acetylation; bromination; DFRC
INTRODUCTION
that pathway b (and/or perhaps c) is involved. Ra-
diotracer/microscopy (Terashima et al., 1993) and sol-
volytic studies (Grabber et al., 1996) suggested that
p-coumarates are attached dominantly to syringyl units
in grass lignins. Thioacidolysis detected p-coumarates
on a maize lignin containing ∼18% p-coumarate (Grab-
ber et al., 1996) but cleaved some esters, lowering its
sensitivity to lignins with low contents of such esters.
DFRC (d erivatization followed by r eductive cleavage)
is a procedure that produces analyzable monomers and
dimers by cleaving R- and â-ethers in lignins (Lu and
Ralph, 1997a,b, 1998a,b, 1999; Peng et al., 1998, 1999;
Ralph and Lu, 1998). One advantage of the method is
that γ-ester groups on lignins remain largely intact (Lu
and Ralph, 1998b). A modified DFRC protocol allowed
successful proof of the occurrence of acetate groups on
lignin side chains in some species (Ralph and Lu, 1998);
kenaf bast fiber lignins, in particular, are extensively
acetylated in nature (Ralph, 1996). Thus, the method
should allow us to confirm that p-coumarate groups are
at the γ-positions of grass lignins and determine their
distribution on syringyl (S) and guaiacyl (G) â-ether
units 8. Three isolated (bamboo, bromegrass, and maize)
lignins were subjected to DFRC degradation to provide
information about sites of p-coumarate attachment and
the likely pathway for its biosynthetic incorporation.
In grasses (Gramineae) hydroxycinnamic acids are
highly involved in the lignification process during plant
cell wall development. Ferulates in ferulate-polysac-
charide esters are intimately incorporated into lignin
via the free-radical coupling processes that typify lig-
nification, producing strong lignin-polysaccharide cross-
linking (Ralph et al., 1995, 1998). Lignins in grasses also
contain p-coumaric acid connected to lignin residues by
ester linkages (Smith, 1955; Nakamura and Higuchi,
1976, 1978a,b; Ralph and Helm, 1993). Elucidation of
the sites of p-coumarate attachment to lignin is impor-
tant to understand mechanisms by which p-coumaric
acid is incorporated into lignins. Three pathways (Fig-
ure 1) could be responsible for acylation of lignins by
p-coumaric acid resulting in two different regiochemis-
tries: (a) free p-coumaric acid 10 reacts with quinone
methide lignin intermediates 4, forming lignin acylated
by p-coumarate at the R-positions of the lignin side
chains 9; (b) preformed hydroxycinnamyl p-coumarates
2 are incorporated by free-radical coupling into lignins
during lignification so that p-coumarates are exclusively
on the γ-positions of lignin side chains, 7 and 8; and (c)
acylation by an activated p-coumarate 11 (e.g., p-
coumaroyl-SCoA) occurs postlignification, resulting in
esters at R- (9) and/or γ-positions (7, 8), depending on
the selectivity of the (presumably required) transferase.
EXPERIMENTAL PROCEDURES
NMR studies on isolated lignins from maize (Ralph
et al., 1994), wheat (Crestini and Argyropoulos, 1997),
and many other grasses including bamboo (Ralph,
unpublished data) reveal that p-coumarates are exclu-
sively at the γ-positions of lignin side chains, suggesting
Gen er a l. All reagents were from Aldrich (Milwaukee, WI)
and used as supplied. Solvents (AR grades) were from Mallinck-
1
rodt (Paris, KY). H, 13C, and 2D NMR (gradient HMQC and
HMBC) spectra were taken on a Bruker DRX-360 instrument
1
fitted with a 5-mm H/broadband gradient probe with inverse
geometry (proton coils closest to the sample). The conditions
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). Carbon/proton designations are based on
conventional lignin numbering (Figure 1).
* 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].
10.1021/jf981140j CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/06/1999

p-Coumarates on Lignins by DFRC
J. Agric. Food Chem., Vol. 47, No. 5, 1999 1989
F igu r e 1. Three possible pathways for incorporation of p-coumarates into lignins. Because >95% of lignification arises from
coupling of a monolignol 1 with a preformed oligolignol 3, monolignol dimerization is neglected here. (a) Nucleophilic addition of
free p-coumaric acid 10 to a quinone methide 4 (formed by coupling of a monolignol 1, at â, with an oligolignol 3, at -O-4, -1, or
-5; weak external nucleophiles (such as p-coumaric acid 10) cannot compete with internal ring closure (such as 4 f 5), so only
quinone methides 4 formed by â-1- or â-O-4- (and not â-5- or â-â-) coupling can give R-esters 9. (b) Incorporation of preformed
hydroxycinnamyl p-coumarates 2 into lignin via free-radical coupling giving acylated lignins 7 and 8; any â-coupling product of
2 (â-O-4, â-5, or â-1) is possible for this pathway. (c) Postlignification acylation by an activated p-coumarate 11 (via a transferase);
this pathway can acylate â-O-4-, â-1-, and â-5-linked units at γ (to give 7 and 8), but only â-O-4- and â-1-linked units at R (to give
9).
Isola ted Lign in s. Lignins were isolated from bamboo
(Bambusa), bromegrass (Bromus), and maize (Zea mays)
according to described methods (Bjo¨rkman, 1954; Ralph and
Hatfield, 1991; Ralph et al., 1994, 1995). Plant material was
ground in a Wiley mill (1-mm screen), and soluble phenolics,
carbohydrates, and other components were removed by suc-
cessive extractions with water, methanol, acetone, and chlo-
roform. The ground wood was then ball-milled, treated with
crude cellulases to degrade most of the polysaccharides, and
extracted with 96:4 dioxane/water. Saccharides and metal ions
were removed from the lyophilized crude lignin using 5 mM
(pH 8) EDTA (Ralph et al., 1994).
Th e DF RC P r oced u r e. The DFRC method is described in
part 1 of this series (Lu and Ralph, 1997b) and in the initial
protocol (Lu and Ralph, 1997a), but 4,4′-ethylidenebisphenol
(Aldrich Chemical Co.) was used as the internal standard.
Response factors for monomers P , G, and S were 1.76, 1.37,
and 1.46, respectively. [P is the DFRC monomeric product from
p-coumaryl units, 4-acetoxycinnamyl acetate, not otherwise
mentioned in this paper but included here for completeness.]
Response factors for the acylated moieties, 4-acetoxy-3-meth-
oxycinnamyl 4-acetoxyphenylpropionate 13a and 4-acetoxy-
3,5-dimethoxycinnamyl 4-acetoxyphenylpropionate 13b, were
2.58 and 3.00, respectively. Molar yields were calculated on
the basis of molecular weights of (underivatized) P )150, G
)180, and S )210; acetates 13a ) 412 and 13b ) 442.
GC a n d GC/MS. The monomers from degraded lignins were
determined by GLC (Hewlett-Packard 5980) using a 0.20-mm
× 25-m DB-1 (J &W Scientific, Folsom, CA) column and a flame
ionization detector with He as carrier gas (10 cm3/min). GC
conditions for monomers were as follows: initial column
temperature, 60 °C, held for 1 min, ramped at 6 °C/min to 300
°C, held for 15 mim; injector temperature, 250 °C; FID detector
temperature, 300 °C. For 13a and 13b, GC conditions were
as follows: initial column temperature, 100 °C, held for 1 min,
ramped at 15 °C/min to 300 °C, held for 25 min; injector
temperature, 250 °C; FID detector temperature, 300 °C. EI-
MS data were collected on a Hewlett-Packard 5970 mass-
selective detector with the same type of column using the same
temperature program.
Syn th esis. Syntheses of 4-acetoxy-3-methoxycinnamyl 4-hy-
droxyphenylpropionate (13a ) and 4-acetoxy-3,5-dimethoxycin-
namyl 4-hydroxyphenylpropionate (13b) were accomplished
according to published methods (Lu and Ralph, 1998c) using
4-hydroxyphenylpropionic acid, Figure 3. 13a was a pale
yellow oil: δH 2.22 (6H, s, OAc), 2.68 (2H, t, J ) 7.5 Hz, P-8),
2.94 (2H, t, J ) 7.5 Hz, P-7), 3.85 (3H, s, OMe), 4.70 (2H, dd,
J ) 6.4, 1.4 Hz, G-γ), 6.32 (1H, dt, J ) 15.8, 6.4 Hz, G-â), 6.65
(1H, dt, J ) 16.0, 1.4 Hz, G-R), 7.00 (2H, m, G-5/G-6), 7.01
(2H, m, P-3/5), 7.18 (1H, s, G-2), 7.27 (1H, s, P-2/6); δC 30.8
(P-8), 36.2 (P-7), 56.2 (OMe), 65.2 (G-γ), 111.2 (G-2), 110.0 (G-
6), 122.4 (P-2/6), 123.7 (G-5), 124.8 (G-â), 130.0 (P-3/5), 133.7
(G-R), 136.20 (G-1), 139.0 (P-1), 140.8 (G-4), 150.3 (P-4), 152.4
(G-3), 169.0 (P-OAc), 169.7 (G-OAc), 172.7 (P-9). 13b was a
clear oil: δH 2.21(3H, s, S-OAc), 2.22 (3H, s, P-OAc), 2.68 (2H,
t, J ) 7.6 Hz, P-8), 2.94 (2H, t, J ) 7.6 Hz, P-7), 3.82 (6H, s,
OMe’s), 4.70 (2H, dd, J ) 6.4, 1.3 Hz, S-γ), 6.35 (1H, dt, J )
15.8, 6.4 Hz, S-â), 6.63 (1H, dt, J ) 16.0, 1.2 Hz, S-R), 6.82
(2H, s, S-2/6), 7.0 (2H, m, P-3/5), 7.27 (2H, s, P-2/6); δC 30.9
(P-8), 36.2 (P-7), 56.5 (OMe’s), 65.2 (S-γ), 104.2 (S-2/6), 122.4
(P-2/6), 124.9 (S-â), 129.1 (S-1), 130.1 (P-3/5), 134.2 (S-R), 139.1
(P-1), 135.7 (S-4), 150.3 (P-4), 153.4 (S-3/5), 168.5 (P-OAc),
169.7 (S-OAc), 172.7 (P-9).
â-Aryl ether models 17 were prepared according to the
procedure of Helm and Ralph (1993). NMR data are deposited
in the NMR Database of Lignin and Cell Wall Model Com-
pounds (Ralph et al., 1999).

1990 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Lu and Ralph
F igu r e 2. Pathway for formation of analyzable ester conjugates 13 from acylated â-aryl ether units 8 in lignins following DFRC.
F igu r e 3. Synthesis of 4-acetoxycinnamyl 4-acetoxyphenylpropionates 13.
RESULTS AND DISCUSSION
When DFRC was first developed, lignins representing
softwoods, hardwoods, dicotyledons, and grasses were
used for testing its capabilities. Chromatograms of
DFRC products from maize and bamboo lignins showed
peaks in the dimeric compound region that were not
present in products from other lignins. From NMR
studies, maize lignin contains large amounts of p-
coumarate exclusively at the γ-positions of lignin side
chains (Figure 1, lignin fragments 7 and 8). The new
dimeric compounds were therefore suspected to be
dimeric degradation products containing p-coumarate
(or a derived product). Some p-coumarate ester linkages
to lignin survived thioacidolysis of the same material,
although results were varied and poorly reproducible
(Grabber et al., 1996). Because DFRC does not affect
lignin γ-esters in models, it seemed reasonable that this
method could detect and determine p-coumarates at-
tached to the γ-positions of â-aryl ether units in lignin
(Figure 2, lignin fragment 8). It should be noted that
the method explored here cannot be used to gain any
information about R-esters because they would be
cleaved in the AcBr step of DFRC. However, such
cleavage could form the basis of a method to detect and
F igu r e 4. â-Aryl ether γ-p-coumarate ester models used for
DFRC release studies.
quantify R-esters plus R-ethers of p-coumaric acid (Lu
and Ralph, 1998b).
Studies with â-aryl ether models 17 (Figure 4) tested
the efficiency of DFRC for releasing hydroxycinnamyl
p-coumarate derivatives and allowed identification of
their degradation products. The â-aryl ether linkages
in models 17 were cleaved in ∼60-65% yield, lower than
the 95% yields from normal unacylated â-aryl ether
models (Lu and Ralph, 1997b). The major product from
17b was sinapyl 4-acetoxyphenylpropionate 13b; no de-

p-Coumarates on Lignins by DFRC
J. Agric. Food Chem., Vol. 47, No. 5, 1999 1991
F igu r e 5. GC-FID chromatograms of DFRC dimers from bamboo and maize lignins. Products 13a and 13b from â-aryl ether
γ-p-coumarate esters 8 are well resolved and easily detected. Their mass spectra are diagnostic. c ) cis, t ) trans.
Ta ble 1. Yield s of Mon om er s a n d Acyla ted Mon om er s
fr om DF RC of Ba m boo, Ma ize, a n d Br om egr a ss Lign in s
affect the release of syringyl or guaiacyl units from
â-aryl ethers. Preferential acylation of syringyl units by
p-coumarate before their incorporation into lignin is
therefore likely. Whole-cell-wall samples were not used
in this study but are expected to produce similar results,
although some fractionation of the p-coumarate into the
extractable lignins has been noted (Ralph et al., 1994).
Whole-cell-wall studies will be aided by solid-phase
extraction cleanup steps currently under development.
Considering the accessibility difference for acylation
of an R-OH versus that of a γ-OH, selective enzymatic
acylation of γ-OH’s by p-coumarates postlignification
would explain why no R-ester has been detected by
NMR. However, this cannot explain why syringyl â-aryl
ether units are far more esterified by p-coumarate than
guaiacyl â-aryl ethers; any γ-acylation should be more
sensitive to the S/G nature of the â-O-aryl unit than to
the unit detected as bearing the p-coumarate. The
observation (Ralph et al., 1994) that p-coumarates on
grass lignins are on many types of units (both isomers
of â-O-4 units, â-5 units, and even cinnamyl alcohol end
groups) suggests that enzyme-assisted acylation of the
lignin polymer (pathway c) is unlikelysthe enzyme
would have to be remarkably nonspecific. The selectivity
required could easily be expected from a transferase
acylating sinapyl versus coniferyl alcohol with an
activated p-coumaric acid. Evidence is therefore mount-
ing that the pathway leading to preferential acylation
is b (Figure 1), in which sinapyl alcohol is preacylated
by p-coumarate and laid down in the cell wall late in
the lignification process when sinapyl alcohol appears
most predominantly (Terashima et al., 1986a,b, 1993;
Terashima and Fukushima, 1988; He and Terashima,
1990; Chabbert et al., 1994a,b). Pathway b was the
pathway originally proposed by Nakamura and Higuchi
(1978a).
monomers (µm/g) p-coumarate products (µm/g)
sample
G
S
S/G
13a
13b
13b/13a
bamboo lignin
maize lignin
bromegrass lignin 454 327 0.72
504 222 0.44
342 124 0.36
6
15
32
5.33
13.73
206
nda
db
a
b
nd, not detected. d, detected, by GC/MS.
esterified monomer was detected. Coniferyl 4-acetoxy-
phenylpropionate (13a ) was obtained from model 17a .
Minor amounts of coniferyl/sinapyl p-coumarates found
by GC/MS were not included in the yield. Compounds
13a and 13b were identified by GC/MS and comparison
of their retention times with those of genuine com-
pounds, which were synthesized (Figure 3) and authen-
ticated by NMR. Compound 13b was a major component
from ether extracts of DFRC degradation products of
maize lignin, as confirmed by 2D NMR experiments.
Mass spectra of 13a and 13b were diagnostic (Figure
5). In each, the molecular ion was observed, and even-
massed ion radicals from one phenolic acetate loss [(M
- 42)+] were abundant. The most abundant ion frag-
ment with m/z 107 is characteristic of the 4-hydroxy-
benzyl moiety from ring P (following loss of ketene from
the acetate). Coniferyl and sinapyl alcohol fragment ions
(m/z 180, 210) are prominent, and other fragment ions
are logical.
Syntheses of 13a and 13b allowed measurement of
response factors so that p-coumarates in lignins released
by DFRC could be quantified by GC. Table 1 shows
DFRC results from lignins isolated from bamboo, maize,
and bromegrass. In bromegrass, the amounts of esters
were too low, but selected-ion mass spectrometry identi-
fied the sinapyl p-coumarate product 13b; 13a could not
be detected. Evidently syringyl units of bamboo and
maize lignins are acylated by p-coumarate to a much
greater degree than guaiacyl units. In bamboo and
maize, the S/G ratios in the esters were 12 and 38 times
greater than those of the normal monomers released by
DFRC. More syringyl units than guaiacyl units are
linked by labile â-aryl ethers, but this would explain
only a ∼2-fold difference in S/G molar ratios of DFRC
products from both lignins. Studies with model com-
pounds showed that acylation did not differentially
CONCLUSIONS
Because DFRC leaves lignol γ-esters essentially in-
tact, it is valuable for identifying esterified lignin
components and for determining the sites of acylation.
This study confirmed that grass lignins are acylated at
the γ-position by p-coumarates; primarily syringyl units
are acylated in maize, bamboo, and bromegrass. Of

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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 F981140J