rearomatizes to afford syringaresinol 2aa by internal trapping
via the two 9-OH groups.13‡ When the 9-OH group is acylated,
as in the “dimerization” of sinapyl p-hydroxybenzoate 1b,
however, it is obviously incapable of trapping the quinone
methide. Consequently, one quinone methide rearomatizes
by external water addition, as is typically seen following
8-O-4-dehydrodimerization.13 At this point there is an internal
OH capable of trapping the other quinone methide moiety.
The resulting product is therefore not a dehydrodimer, but
the product 2bb, with a molecular mass 16 units higher. The
cross-coupling reaction between 1a and 1b also produces an
intermediary bis-quinone methide, but one such moiety is
internally trapped by the single 9-OH. Rearomatization of
the other requires water addition, and produces product 2ab.
Finding the cross-coupling product 2ab in plants is compelling
evidence that sinapyl p-hydroxybenzoate must therefore be
an authentic “monomer” in the lignin (or lignan) pathway;
it is unlikely to have arisen from the dehydrodimer of sinapyl
alcohol, 2aa.
Indeed product 2ab was identified in the actively lignifying
xylem tissue of 3 month old poplar.9 The product synthesized
here was shown to be identical by its UV and mass (including
MS-MS) spectral data, and HPLC retention time (including
by co-injection). Its detection in actively lignifying tissue
suggests that it may be a compound destined for the lignin
polymer. More importantly, it demonstrates that sinapyl
p-hydroxybenzoate is the “monomer” in the coupling reac-
tion in planta. A p-hydroxybenzoyl transferase is therefore
implicated for acylating the monolignol 1a to feed the acylated
substrate 1b into the lignification process in poplar, where the
lignin contains p-hydroxybenzoylated units.15 The product
2ab has also been isolated previously, without comment, from
Salix sachalinensis.16
Notes and references
† Synthesis of the cross-coupled product from sinapyl alcohol and
sinapyl p-hydroxybenzoate: sinapyl p-hydroxybenzoate5 (700 mg,
2.12 mmol) and sinapyl alcohol12 (460 mg, 2.19 mmol) were dissolved
in acetone (100 ml) and added into phosphate buffer (20 mM sodium
dihydrogen phosphate, pH 4.5, 400 ml) in 500 ml flask. Horseradish
peroxidase (Sigma, Type II, 150–250 units mg−1, 8 mg) was added
followed by addition of the hydrogen peroxide urea complex (200 mg,
2.13 mmol) dissolved in 10 ml water. This mixture was stirred for 1 h
when TLC indicated complete starting material disappearance. The
mixture was saturated with NH4Cl and acidified with dilute aqueous
HCl (3%) to pH around 3–4 and extracted with EtOAc (300 ml × 2). The
organic phase was dried over MgSO4 and filtered. The crude product
(pale white foam) was obtained after evaporation of the organic phase.
The cross-coupled compound 2ab (280 mg, 25% yield) was isolated
by silica gel chromatography with cyclohexane–EtOAc, 1:1 as eluting
solvent. UV and MS data have been reported separately.9 NMR
(acetone-d6) dH 4.96 (1H, d, J = 5.0 Hz, A7), 3.99 (1H, m, A8), 4.14
(2H, m, A9s), 3.78* (6H, s, A3/5-OMe), 6.72 (2H, s, A2/6); 4.90 (1H,
d, J = 6.3 Hz, B7), 2.55 (1H, m, B8), 4.41 (1H, m, B9a), 4.68 (1H, m,
B9b), 3.74* (6H, s, B3/5-OMe), 6.66 (2H, s. B2/6); 7.77 (2H, m, C2/6),
6.87 (2H, m, C3/5) (* assignments may be interchanged); dC 136.0 (A1),
104.53 (A2/6), 148.5 (A3/5), 135.9 (A4), 72.6 (A7), 48.4 (A8), 70.0 (A9);
134.5 (B1), 104.45 (B2/6), 148.5 (B3/5), 136.0 (B4), 85.3 (B7), 49.8 (B8),
64.0 (B9); 122.4 (C1), 132.5 (C2/6), 116.0 (C3/5), 162.6 (C4), 166.4 (C7).
Small amounts of other products such as 8-O-4-dehydrodimers and
aryltetralin 8-8-products were not further examined.
‡ In reactions with sinapyl alcohol alone, the 8-O-4-dehydrodimer is
also produced but typically at less than 9% yield.14
§ QMbb: NMR (DMSO-d6) dH 3.66 (3H, s, S3-OMe), 3.70 (3H, s, S5-
OMe), 4.02 (1H, m, S8), 4.42 (2H, m, S9), 6.56 (1H, d, J = 10.3, S7), 6.56
(1H, d, J = 1.9, S6), 6.82 (2H, m, P3/5), 6.93 (1H, d, J = 1.9, S2), 7.82
(2H, m, P2/6); dC 40.7 (S8), 55.55 (S5-OMe), 55.62 (S3-OMe), 65.9 (S9),
104.8 (S6), 112.9 (S2), 115.9 (P3/5), 121.1 (P1), 132.3 (P2/6), 133.5 (S1),
140.4 (S7), 152.2 (S5), 153.7 (S3), 163.3 (P4), 166.2 (P7), 175.29 (S4).
1 W. Boerjan, J. Ralph and M. Baucher, Lignin Biosynthesis, Annu.
Rev. Plant Biol., 2003, 54, 519.
One of the more intriguing and unexpected chemical aspects
of the homo-coupling reaction using sinapyl p-hydroxybenzoate
1b in peroxidase–H2O2 at pH 5 to independently generate
compound 2bb was that the intermediary quinone methide
QMbb was isolable and stable.§ A pale yellow precipitate
formed that was difficult to dissolve in common organic
solvents. NMR in DMSO-d6 revealed it to be the quinone
methide QMbb. Although certain syringyl and guaiacyl
quinone methides were sufficiently stable in solution to allow
NMR spectra to be recorded in 1983,17 they have generally been
considered unstable, although crystalline bromo analogs have
been long known.18 QMbb in the solid state did not degrade
after over 8 months in a freezer. Regrettably, it has not yet been
possible to obtain sufficiently good crystals for an X-ray crystal
structure. Extracting the entire reaction product into EtOAc
and washing with saturated aqueous NH4Cl acidified with HCl
produces mainly compound 2bb (along with the tetrahydro-
naphthalene 2bb) demonstrating that compound 2bb derives
from this quinone methide intermediate.
2 J. Ralph, K. Lundquist, G. Brunow, F. Lu, H. Kim, P. F. Schatz,
J. M. Marita, R. D. Hatfield, S. A. Ralph, J. H. Christensen and
W. Boerjan, Lignins: natural polymers from oxidative coupling of
4-hydroxyphenylpropanoids, Phytochem. Rev., 2004, 3, in press.
3 J. C. del Rio, A. Gutierrez and A. T. Martinez, Identifying
acetylated lignin units in non-wood fibers using pyrolysis-gas
chromatography/mass spectrometry, Rapid Commun. Mass
Spectrom., 2004, 18, 1181.
4 J. Ralph and F. Lu, The DFRC method for lignin analysis. Part 6.
A modified method to determine acetate regiochemistry on native
and isolated lignins, J. Agric. Food Chem., 1998, 46, 4616.
5 Y. Nakamura and T. Higuchi, Ester linkage of p-coumaric acid
in bamboo lignin. II. Syntheses of coniferyl p-hydroxybenzoate
and coniferyl p-coumarate as possible precursors of aromatic acid
esters in lignin, Cellul. Chem. Technol., 1978, 12, 199.
6 F. Lu and J. Ralph, Preliminary evidence for sinapyl acetate as a
lignin monomer in kenaf, Chem. Commun., 2002, 90.
7 J. Ralph, R. D. Hatfield, S. Quideau, R. F. Helm, J. H. Grabber and
H.-J. G. Jung, Pathway of p-coumaric acid incorporation into maize
lignin as revealed by NMR, J. Am. Chem. Soc., 1994, 116, 9448.
8 F. Lu and J. Ralph, Detection and determination of p-coumaroylated
units in lignins, J. Agric. Food Chem., 1999, 47, 1988.
In conclusion, demonstration that sinapyl p-hydroxybenzoate
1b is an authentic precursor of lignification augments the
finding that sinapyl acetate is implicated in kenaf bast fiber
lignification6 and the evidence for sinapyl and coniferyl
p-coumarate in maize (and other grasses).7,8 The mechanism
therefore appears to be a general one for the three types of
natural lignin acylation observed in nature. Future research will
be aimed at determining if low levels of structures resulting
from incorporation of 2ab and 2bb can be detected in lignins.
The ultimate goals will be to find the transferases and their
genes, and to attempt to understand the role of lignin acylation
in these plants.
9 K. Morreel, J. Ralph, H. Kim, F. Lu, G. Goeminne, S. A. Ralph,
E. Messens and W. Boerjan, Profiling of oligolignols reveals
monolignol coupling conditions in lignifying poplar xylem, Plant
Physiol., 2004, in press.
10 K. Morreel, J. Ralph, F. Lu, G. Goeminne, R. Busson, P. Herdewijn,
J. L. Goeman, J. Van der Eycken, W. Boerjan and E. Messens,
Phenolic profiling of COMT-deficient poplar reveals novel
benzodioxane oligolignols, Plant Physiol., 2004, in press.
11 K. Syrjanen and G. Brunow, Regioselectivity in lignin biosynthesis.
The influence of dimerization and cross-coupling, J. Chem. Soc.,
Perkin Trans. 1, 2000, 183.
12 F. Lu and J. Ralph, Highly selective syntheses of coniferyl and
sinapyl alcohols, J. Agric. Food Chem., 1998, 46, 1794.
13 K. Freudenberg and A. C. Neish. Constitution and Biosynthesis of
Lignin, Springer-Verlag, Berlin–Heidelberg–New York, 1968.
14 M. Tanahashi, H. Takeuchi and T. Higuchi, Dehydrogenative
polymerization of 3,5-disubstituted p-coumaryl alcohols, Wood
Res., 1976, 61, 44.
Acknowledgements
We gratefully acknowledge partial funding through the DOE
Energy Biosciences program (#DE-AI02-00ER15067) and
the USDA-CSREES National Research Initiatives (Improved
Utilization of Wood and Wood Fiber #2001-02176).
15 H. Meyermans, K. Morreel, C. Lapierre, B. Pollet, A. De Bruyn,
R. Busson, P. Herdewijn, B. Devreese, J. Van Beeumen, J. M. Marita,
J. Ralph, C. Chen, B. Burggraeve, M. Van Montagu, E. Messens
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 8 8 8 – 2 8 9 0
2 8 8 9