attack of the 9-OH on the quinone methide intermediate QM2,
Scheme 1. The 9-acetylation prevents such a reaction. Other
pathways must therefore be in effect producing other products.
The important point is that the acetyl group can remain attached
in non-resinol 8–8-coupling products, products that could not
have arisen from post-coupling acetylation reactions. {We
recognize that there remains the remote possibility, not ruled out
by the observations presented here, that processes involving
subsequent opening of resinols 3 and acetylation of the opened
products could occur, but that would appear to require at least
two enzymatic activities. A resinol ring opening and reduction
is effected, for example, by NADPH-dependent pinoresinol–
lariciresinol reductase.10}
In seeking preliminary evidence for sinapyl acetate in-
corporation, it didn’t appear necessary to elucidate the full
coupling and cross-coupling pathways for sinapyl acetate. All
that was required was to show that sinapyl acetate 2, in coupling
and cross-coupling reactions, would give acetylated products
that would produce DFRCA 8–8-linked products identical to
those that are released from kenaf (lignins) and not from plants
having non-acetylated lignins. Oxidation of sinapyl alcohol 1
with H2O2/peroxidase or metal oxidants typically gives the
lignan syringaresinol 3 as the predominant dehydrodimeric
product, along with a small amount of the 8–O–4-coupled
product.11 In this study, sinapyl alcohol was oxidized with
H2O2–peroxidase in a 20 mM buffer solution containing 20%
acetone; syringaresinol 3 was the only 8–8-product, produced in
over 90% yield. DFRCA treatment of syringaresinol 3 yielded
aryltetralin 6a, Scheme 1, as a major product.† Similar
compounds are produced following thioacidolysis of
8–8-linked lignin units.12 Oxidation of sinapyl acetate 2
(Scheme 1) under similar conditions yielded a mixture of
currently uncharacterized compounds retaining acetate groups.
DFRCA treatment of the mixture yielded 6c, the diacetate analog
of 6a (from sinapyl alcohol). The structural analogy, from MS
spectra (Fig. 1), indicates that sinapyl acetate also undergoes
8–8-coupling, and that at least one of the products 5 (Scheme 1,
although the exact structure has not yet been determined)
produces the aryltetralin 6c following DFRCA treatment.
Oxidation of a mixture of sinapyl alcohol 1 and sinapyl acetate
2 must result in cross-coupling reactions to produce crossed
8–8-coupled structures 4, since DFRCA degradation now
produces mono-acetylated aryltetralins 6b in addition to the
non- and di-acetylated analogs 6a and 6c (as evidenced by GC-
MS).
participates in formation of its lignin. Selected ion chromato-
grams of TLC-fractionated DFRCA products from kenaf lignin
(Fig. 1) or whole kenaf cell walls clearly show the presence of
all three DFRCA products, compounds 6a–c. Comparison of GC
retention times and mass spectra of DFRCA products with those
from the in vitro coupling reactions of sinapyl alcohol and
sinapyl acetate indicates that compounds 6b and 6c derive from
sinapyl acetate. Compound 6a derives from normal lignins, but
the acetylated analogues 6b and 6c do not.
Although a great deal remains to be done to detail the
coupling reactions, authenticate the nature and stereochemistry
of the products, and fully elucidate their DFRCA products, the
preliminary data presented here appears to us to provide rather
compelling evidence that sinapyl acetate is involved in
lignification in kenaf, and is the likely source of the high
9-acetylation observed in kenaf bast fiber lignins. Detection of
9-acetates in syringyl 8–8-coupled DFRCA products 6b–c from
kenaf suggests the existence of substructures which are likely
formed from dehydrogenative coupling of sinapyl acetate itself
or cross-coupling with sinapyl alcohol during the lignification
process and provides evidence that acetates on kenaf lignin are
formed through incorporation of sinapyl acetate, as a lignin
precursor, into lignin macromolecules by radical coupling.
Sinapyl acetate therefore appears to be an authentic lignin
monomer in kenaf.
The preliminary evidence provided here that acylation is at
the monolignol stage allows researchers to seek the substrates
and presumed transferases involved in the specific acylation of
monolignols, and to identify the genes responsible, allowing the
process to be genetically manipulated.
We are grateful to USDA-NRI for partial funding
(#2001-02176 in the Improved Utilization of Wood and Wood
Fiber section), and to James Han (U.S. Forest Products Lab.,
Madison, WI) for providing kenaf samples.
Notes and references
† In addition to the MS data in Fig. 1, NMR (360 MHz, acetone-d6) was
diagnostic: dH 1.06, 1.10, 1.17, 1.18 (4 3 3H, t, J = 7.5 Hz, propyl-Me),
2.03 (1H, m, B8), 2.06 (3H, s, B-OAc), 2.27 (1H, m, A8), 2.30, 2.38, 2.54,
2.56 (4 3 2H, q, J = 7.5 Hz, propyl-CH2), 2.82 (2H, m, 7), 3.25 (3H, s, B3-
OMe), 3.69 (6H, s, A-OMe’s), 3.80 (3H, s, B5-OMe), 4.00 (1H, m, A9a),
4.13 (1H, m, B9a), 4.21 (1H, d, J = 6.6 Hz, A7), 4.21 (1H, m, B9b), 4.30
(1H, m, A9b), 6.45 (2H, s, A2/6), 6.71 (1H, s, B2). The parent compound,
8-(4-hydroxy-3,5-dimethoxyphenyl)-6,7-bis-hydroxymethyl-1,3-dimeth-
oxy-5,6,7,8-tetrahydronaphthalen-2-ol, has been isolated from several plant
sources according to Beilstein; the earliest reference appears to be by
Weinges.13
It would be reasonable to expect that substructures 4 and 5
exist (in phenol-etherified form) in kenaf if sinapyl acetate
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Fig. 1 Mass spectra and selected-ion chromatograms of DFRCA products
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1 and 2 acetates.
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