Cross-coupling of hydroxycinnamyl aldehydes into lignins

Article abstract of DOI:10.1021/ol005906o

(equation presented) Pathways for hydroxycinnamyl aldehyde incorporation into lignins are revealed by examining transgenic plants deficient in cinnamyl alcohol dehydrogenase, the enzyme that converts hydroxycinnamyl aldehydes to the hydroxycinnamyl alcoho

Full text of DOI:10.1021/ol005906o

ORGANIC
LETTERS
2000
Vol. 2, No. 15
2197-2200
Cross-Coupling of Hydroxycinnamyl
Aldehydes into Lignins
Hoon Kim,†,‡ John Ralph,*,†,‡ Nabila Yahiaoui,§ Michel Pean,| and
Alain-M. Boudet§
U.S. Dairy Forage Research Center, USDA-Agricultural Research SerVice,
Madison, Wisconsin 53706, Department of Forestry, UniVersity of Wisconsin-Madison,
Madison, Wisconsin 53706, Poˆle de Biotechnologie Ve´ge´tale, Unite´ Mixte de
Recherche, F31326 Castanet Tolosan, France, and Groupe de Recherches Applique´es
en Phytotechnologie, Commissariat a` l’Energie Atomique/Cadarache, DSV-DEVM,
Saint-Paul-Lez-Durance Cedex, France
jralph@facstaff.wisc.edu
Received April 6, 2000
ABSTRACT
Pathways for hydroxycinnamyl aldehyde incorporation into lignins are revealed by examining transgenic plants deficient in cinnamyl alcohol
dehydrogenase, the enzyme that converts hydroxycinnamyl aldehydes to the hydroxycinnamyl alcohol lignin monomers. In such plants the
aldehydes incorporate into lignins via radical coupling reactions. As diagnostically revealed by long-range 13C−1H correlative NMR, sinapyl
aldehyde (3,5-dimethoxy-4-hydroxy-cinnamaldehyde) 8-O-4-cross-couples with both guaiacyl (3-methoxy-4-hydroxyphenyl-propanoid) and syringyl
(3,5-dimethoxy-4-hydroxyphenyl-propanoid) units, whereas coniferyl aldehyde cross-couples only with syringyl units.
Lignins are polymeric natural products present in large
quantities in the cell walls of terrestrial plants. They arise
principally from one or more of the three monolignols,
p-coumaryl (4-hydroxycinnamyl), coniferyl (4-hydroxy-3-
methoxy-cinnamyl), and sinapyl (3,5-dimethoxy-4-hy-
droxycinnamyl) alcohols, analogues varying in their degrees
of methoxylation.1 However, many other phenolic compo-
nents also intimately incorporate into lignins;2 acylated
hydroxycinnamyl alcohols3 and hydroxycinnamate esters4 are
notable examples. Various monolignol precursors including
hydroxycinnamyl aldehydes5 and 5-hydroxyconiferyl alcohol6
are incorporated into lignins, particularly when the plant is
deficient in certain enzymes on the monolignol biosynthetic
H.; Jimenez-Monteon, G.; Zhang, Y.; Jung, H.-J. G.; Landucci, L. L.;
MacKay, J. J.; Sederoff, R. R.; Chapple, C.; Boudet, A. M. In Progress in
Lignocellulosics Characterization; Argyropoulos, D. S., Rials, T., Eds.;
TAPPI Press: Atlanta, GA, 1999; pp 55-108. (d) Whetten, R. W.; MacKay,
J. J.; Sederoff, R. R. Annu. ReV. Plant Physiol. Plant Mol. Biol. 1998, 49,
585-609.
(3) Acetylated monolignols: (a) Ralph, J.; Lu, F. J. Agric. Food Chem.
1998, 46, 4616-4619. (b) Ralph, J. J. Nat. Prod. 1996, 59, 341-342.
p-Coumaroylated monolignols: (c) Lu, F.; Ralph, J. J. Agric. Food Chem.
1999, 47, 1988-1992. (d) Ralph, J.; Hatfield, R. D.; Quideau, S.; Helm,
R. F.; Grabber, J. H.; Jung, H.-J. G. J. Am. Chem. Soc. 1994, 116, 9448-
9456.
(4) (a) Ralph, J.; Hatfield, R. D.; Grabber, J. H.; Jung, H. G.; Quideau,
S.; Helm, R. F. In Lignin and Lignan Biosynthesis; Lewis, N. G., Sarkanen,
S., Eds.; American Chemical Society: Washington, DC, 1998; ACS
Symposium Series no. 697, pp 209-236. (b) Ralph, J.; Grabber, J. H.;
Hatfield, R. D. Carbohydr. Res. 1995, 275, 167-178.
† USDA-Agricultural Research Service.
‡ University of Wisconsin-Madison.
§ Unite´ Mixte de Recherche.
| Commissariat a` l’Energie Atomique.
(1) (a) Freudenberg, K.; Neish, A. C. Constitution and Biosynthesis of
Lignin; Springer-Verlag: Berlin-Heidelberg-New York, 1968. (b) Harkin,
J. M. In OxidatiVe Coupling of Phenols; Taylor, W. I., Battersby, A. R.,
Eds.; Marcel Dekker: New York, 1967; pp 243-321. (c) Li, L.; Popko, J.
L.; Umezawa, T.; Chiang, V. L. J. Biol. Chem. 2000, 275, 6537-6545.
(2) (a) Boudet, A.-M. Trends Plant Sc. 1998, 3, 67-71. (b) Sederoff, R.
R.; MacKay, J. J.; Ralph, J.; Hatfield, R. D. Curr. Opin. Plant Biol. 1999,
2, 145-152. (c) Ralph, J.; Marita, J. M.; Ralph, S. A.; Hatfield, R. D.; Lu,
F.; Ede, R. M.; Peng, J.; Quideau, S. p.; Helm, R. F.; Grabber, J. H.; Kim,
10.1021/ol005906o CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/28/2000

pathway. Lignin-biosynthetic-pathway mutants and trans-
genics provide useful perturbations to “normal” lignification
that allow normally minor components to be substantially
enhanced and therefore structurally analyzed, to better
understand the lignification process.
a chain-extended oligomer/polymer, also with the general
formula 3. Recent studies have begun to elucidate the
propensity for the monolignols to cross-couple with (free-
phenolic) guaiacyl and syringyl units in lignins.10 No data
on cross-coupling of the hydroxycinnamaldehydes into
lignins are available.
The lignin-biosynthetic-pathway enzyme CAD (cinnamyl
alcohol dehydrogenase) is the last enzyme on the pathway
to the monolignols coniferyl and sinapyl alcohols 2G and
2S, from which lignins are normally derived (Figure 1).
When CAD is downregulated, the hydroxycinnamyl aldehyde
precursors to the monolignols, coniferyl aldehyde 1G and
sinapyl aldehyde 1S, build up and may be incorporated into
the polymer by radical coupling.5 Indeed, it was readily
shown that coniferyl aldehyde would incorporate into
synthetic lignins under biomimetic conditions.5f Hydroxy-
cinnamyl aldehydes 1 and hydroxybenzyl aldehydes 5
derived from them are well-known components of lignins
isolated from normal plants,7 as well as mutant or transgenic
plants.5 In a CAD-downregulated tobacco transgenic, the
aldehyde incorporation into the lignin was significant.5b
Detailed proof that the aldehydes intimately incorporate into
the polymer, cross-coupling with normal lignin oligomers,
will be published elsewhere8 (brief arguments are given
below). In this paper, we report on the cross-coupling
propensities of coniferyl and sinapyl aldehydes with guaiacyl
and syringyl units in lignins. Data revealing such details of
plant lignification are rarely obtained.
With the availability of a 13C-enriched lignin from a CAD-
deficient tobacco transgenic,5b NMR methods can be used
to ascertain hydroxycinnamyl aldehyde in vivo cross-
coupling propensities. Figure 1 (right) shows a selected
region of the HMBC spectrum from the CAD-deficient
tobacco lignin,11 with peaks in the 13C-projection and the
resultant contours in the HMBC spectrum colored to match
structures on the left for easy identification. In fact, the
aldehyde structures in the lignins likely have been further
incorporated into the polymer by primarily 4-O-coupling with
the next monolignol, so they are not strictly the phenolic
compounds 4 indicated. Available coupling sites are indicated
in Figure 1 by the dashed arrows. Correlation of the aldehyde
carbonyl carbons in crossed dimers 4 to the H7-protons
3-bonds away identifies the type of lignin units involved.
Protons H7 resonate at ∼7.3 ppm for hydroxycinnamyl
aldehydes (either 1G or 1S) coupled 8-O-4 to guaiacyl units
3G (compounds 4GG and 4SG, δC ) 188.1 ppm), whereas
they resonate considerably upfield at ∼6.7 ppm when
coupled 8-O-4 to syringyl units 3S (compounds 4GS and
4SS, δC ) 186.8 ppm). Correlations from these H7 protons
into the ring identify the hydroxycinnamyl aldehyde involved
in the coupling; 3-bond correlations identify equivalent S2/6
carbons derived from sinapyl aldehyde 1S units at higher
field than the nonequivalent G2 and G6 carbons from units
derived from coniferyl aldehyde 1G. Therefore, for cross-
coupled units 4 in lignins, the G/S nature of both the
hydroxycinnamyl aldehyde component (coupled 8-) and the
lignin unit (coupled 4-O-) are diagnostically revealed. Other
aromatic protons in the complex lignin polymer resonate in
the H7 regions, so correlations that are not of interest here
will result; an absence of correlations is therefore more
diagnostic, revealing the absence of a component.
Radical cross-coupling of monolignols 2 with the growing
lignin oligomer/polymer 3 is the major reaction occurring
during lignification. Thus the hydroxycinnamyl alcohol 2
(primarily at its â-position) couples with phenolic units 3
(at the 4-O- or 5-position for guaiacyl units 3G, and almost
exclusively at the 4-O-position for syringyl units 3S)9 to form
(5) (a) Ralph, J.; MacKay, J. J.; Hatfield, R. D.; O’Malley, D. M.;
Whetten, R. W.; Sederoff, R. R. Science 1997, 277, 235-239. Note that
the new aldehyde peaks (analogous to 4 in Figure 1) were erroneously
assigned as 2-methoxybenzaldehydes in this paper, as subsequently
corrected.2c,5b (b) Ralph, J.; Hatfield, R. D.; Piquemal, J.; Yahiaoui, N.;
Pean, M.; Lapierre, C.; Boudet, A.-M. Proc. Nat. Acad. Sci. 1998, 95,
12803-12808. (c) Yahiaoui, N.; Marque, C.; Myton, K. E.; Negrel, J.;
Boudet, A.-M. Planta 1998, 204, 8-15. (d) Stewart, D.; Yahiaoui, N.;
McDougall, G. J.; Myton, K.; Marque, C.; Boudet, A. M.; Haigh, J. Planta
1997, 201, 311-318. (e) Halpin, C.; Knight, M. E.; Foxon, G. A.; Campbell,
M. M.; Boudet, A.-M.; Boon, J. J.; Chabbert, B.; Tollier, M.-T.; Schuch,
W. Plant J. 1994, 6, 339-350. (f) Higuchi, T.; Ito, T.; Umezawa, T.; Hibino,
T.; Shibata, D. J. Biotechnol. 1994, 37, 151-158. (g) Provan, G. J.; Scobbie,
L.; Chesson, A. J. Sci. Food Ag. 1997, 73, 133-142. (h) Vailhe, M. A. B.;
Besle, J. M.; Maillot, M. P.; Cornu, A.; Halpin, C.; Knight, M. J. Sci. Food
Agric. 1998, 76, 505-514. (i) Halpin, C.; Holt, K.; Chojecki, J.; Oliver,
D.; Chabbert, B.; Monties, B.; Edwards, K.; Barakate, A.; Foxon, G. A.
Plant J. 1998, 14, 545-553.
(6) (a) Lapierre, C.; Tollier, M. T.; Monties, B. C. R. Acad. Sci., Ser. 3
1988, 307, 723-728. (b) Jouanin, L.; Goujon, T.; de Nada¨ı, V.; Martin,
M.-T.; Mila, I.; Vallet, C.; Pollet, B.; Yoshinaga, A.; Chabbert, B.; Petit-
Conil, M.; Lapierre, C. Plant Physiol. 2000, in press.
(7) (a) Lu¨demann, H.-D.; Nimz, H. Makromol. Chem. 1974, 175, 2409-
2422. (b) Lai, Y. Z.; Sarkanen, K. V. In Lignins, Occurrence, Formation,
Structure and Reactions; Sarkanen, K. V., Ludwig, C. H., Eds.; Wiley-
Interscience: New York, 1971; pp 165-240. (c) Chen, C.-L. In Methods
in Lignin Chemistry; Lin, Y., Dence, W. W., Eds.; Springer-Verlag: New
York, 1992; pp 446-457.
(8) Ralph, J.; Kim, H.; Lu, F.; Ralph, S. A.; Landucci, L. L.; Ito, T.;
Kawai, S.; Ohashi, H.; Higuchi, T. Incorporation of hydroxy-cinnamalde-
hydes into lignins; 2000, in preparation.
Of the four possible aldehyde incorporation products 4,
only three can be detected in the lignin by NMR methods.
Product 4GG is conspicuously absent. The data imply that
sinapyl aldehyde 1S cross-couples with both guaiacyl and
syringyl units (to form cross-coupled structures 4SG and
4SS) but that coniferyl aldehyde 1G cross-couples only with
syringyl units 3S and not with guaiacyl units 3G. Thus cross-
coupling product 4GS is readily detected, whereas 4GG
cannot be detected even when spectra are viewed at close to
the baseplane noise level. Attempting to prepare compounds
modeling 4GG by biomimetically cross-coupling coniferyl
aldehyde 1G (at the 8-position) with coniferyl alcohol 2G
or with a simple guaiacyl model 1-(4-hydroxy-3-methoxy)-
(10) (a) Syrjanen, K.; Brunow, G. J. Chem. Soc., Perkin Trans. 1 1998,
3425-3429. (b) Syrjanen, K.; Brunow, G. J. Chem. Soc., Perkin Trans. 1
2000, 183-187.
(11) The acetylated CAD-deficient transgenic tobacco lignin was that
described previously (ref 5b). Gradient-HMBC spectra of the lignin and
model compounds were run on a Bruker DRX-360 using a 5 mm inverse
{1H-broadband} probe equipped with 3-axis gradients. The long-range
coupling delay was 80 ms.
(9) Another type of coupling is possible from monolignols 2 (at the
â-position) with guaiacyl or syringyl units 3 at the aromatic 1-position (â-
1-coupling); see ref 1.
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Org. Lett., Vol. 2, No. 15, 2000

Figure 1. Cross-coupling of hydroxycinnamyl aldehydes 1G and 1S with guaiacyl and syringyl lignin units 3G and 3S can potentially
produce four 8-O-4-cross-coupled structures 4. Only three of the four cross-products (4GS, 4SG, and 4SS but not 4GG) are significantly
represented, as determined by analysis of the gradient-HMBC partial spectrum to the right. The HMBC section shows, in a lignin isolated
from CAD-deficient tobacco stems, correlations from the aldehyde carbonyl carbons (185-195 ppm) to the H7 protons within 3-bonds in
structures 1 and 4 that have been further incorporated into lignin at positions indicated by the dashed arrows. (For compounds 5, it is H2
and H6, rather than H7, that are 3-bonds from the aldehyde carbonyl carbon). These H7 protons diagnostically correlate with the 2- and
6-carbons of the cinnamyl aldehyde ring; the G2 and G6 carbons for guaiacyl units, and the higher-field equivalent S2/6 carbons are
identified and colored correspondingly. Colored dots on the structures indicate the carbons correlated (by 3JC-H) to the H7-protons (which
are shown in a larger font size) shown in the HMBC spectrum. The aldehydes at ∼188.1 ppm, with their corresponding H7s at ∼7.3 ppm
result from hydroxycinnamyl aldehydes 1 8-O-4-coupled to guaiacyl units 3G in lignin; correlations from H7 indicate, however, that sinapyl
aldehyde 1S, but not coniferyl aldehyde 1G, couples with guaiacyl units 3G; the green X’s show where model 4gg correlations exist.12 The
aldehydes at ∼186.7 ppm, with their corresponding H7s at ∼6.7 ppm result from hydroxycinnamyl aldehydes 1 8-O-4-coupled to syringyl
units 3S in lignin; correlations from H7 indicate that sinapyl aldehyde 1S and coniferyl aldehyde 1G each couple with syringyl units 3S.
Both hydroxycinnamaldehydes 1G and 1S couple at the 4-O-position with monolignols 2 at â to produce structures 1G and 1S in lignin,
as seen by the aldehyde peaks at 193.9 ppm, their correlation with H7-protons at ∼7.6 ppm, and their correlations with both S2/6 and G2
and G6 carbons (colored). Benzyl aldehydes 5, principally syringyl aldehyde 5S, are also 4-O-coupled by monolignols at â (peaks labeled
5). S ) syringyl, G ) guaiacyl, CCR ) cinnamoyl-CoA reductase, CAD ) cinnamyl alcohol dehydrogenase, F5H ) ferulate 5-hydroxylase
(which has recently been renamed CAld-5H ) coniferyl aldehyde 5-hydroxylase, to reflect the preferred in vivo substrate16), OMT )
O-methyl transferase, POD ) peroxidase. Fully authenticated peak assignments will be reported elsewhere.12 Convention for compound
numbering: e.g., 4GS ) a guaiacyl aldehyde (coniferyl aldehyde 1G) coupled 8-O-4 to a syringyl lignin unit (3S).
ethanol (at the 4-O-position), also failed to produce 8-O-4-
coupled cross-products. Apparently the same factors that
promote cross-coupling in vitro apply in vivo, providing new
evidence that radical coupling reactions in lignification are
likely to be under simple chemical control in the plant.2b In
the crossed dimers, the chemical shifts assigned match with
model compounds synthesized by traditional means.12
Examination of the spectra provides significant information
regarding the incorporation of hydroxycinnamyl aldehydes
into “bulk” vs “endwise” polymers.13 Bulk lignins are
characterized by frequent dimerization reactions, whereas
endwise polymerization, the normal process occurring during
lignification in the secondary cell wall, is from cross-coupling
of monomers with the growing polymer. Dimerization of
Org. Lett., Vol. 2, No. 15, 2000
2199

coniferyl aldehyde produces the 8-O-4-coupled dimer (analo-
gous to 4GG, Figure 1), along with the other anticipated
8-8- and 8-5-coupled dimers.14 The absence of signals for
coniferaldehyde coupled with G-units (of which the conifer-
aldehyde dimer 4GG is an example), is evidence that
coniferaldehyde does not dimerize and therefore is not
significantly involved in bulk lignification. Further evidence
will be described elsewhere.8
Also available from the spectrum in Figure 1 is some
information regarding the incorporation of the hydroxycin-
namyl aldehydes 1 and their derived hydroxybenzyl alde-
hydes 5 at ring 4-O-positions. Syringyl aldehyde 5S, as seen
from the contours correlating the aldehyde carbon with the
2/6-protons (which are now the protons within 3-bonds of
the aldehyde carbonyl carbon), appears to be well incorpo-
rated, but there is little evidence for incorporation of vanillin
5G. Syringyl aldehyde 5S can only couple at its 4-O-position
with a monomer radical, not with the growing polymer.
Vanillin 5G can react either 4-O- with a monomer or 5- with
either a monomer or an oligomer 3. The hydroxycinnamyl
aldehydes 1 can cross-couple to give products 4 as described
above and can also couple at their 4-O-positions with
monolignols (at â). Contours correlating the H7-proton in
these units with both 1S-2/6 carbons, as well as 1G-2 and
1G-6 carbons, indicate that both sinapyl aldehyde 1S and
coniferyl aldehyde 1G will incorporate at their 4-O-positions
(by radical coupling with monolignols at Câ). NMR does
not readily reveal information regarding the nature of the
cross-coupling partner in this case.
In summary, in 8-O-4-coupling reactions, coniferyl alde-
hyde cross-couples only with syringyl units,15 whereas
sinapyl aldehyde cross-couples with both guaiacyl and
syringyl units. Syringyl aldehyde incorporates efficiently into
lignins, whereas vanillin units are either not produced
extensively or are incorporated poorly into 4-O-structures.
Sinapyl and coniferyl aldehydes 1 both appear to 4-O-
incorporate by radical coupling with monolignols. These
observations require no departure from the existing theory
of radical coupling of phenols into polymeric lignins since
they appear to reflect simple chemical coupling propensities.
(12) Model compounds for the hydroxycinnamaldehyde-lignin cross-
coupled products were synthesized following normal lignin â-O-4 model
pathways (Nakatsubo, F.; Sato, K.; Higuchi, T. Holzforschung 1975, 29,
165-168). Elimination from the â-hydroxyester 6, as previously published
(Ralph, J.; Quideau, S.; Grabber, J. H.; Hatfield, R. D. J. Chem. Soc., Perkin
Trans. 1 1994, 3485-3498) accompanied by debenzylation, DIBAL
reduction of the ester to the alcohol (Quideau, S.; Ralph, J. J. Agric. Food
Chem. 1992, 40, 1108-1110.), and final allylic oxidation with 2,3-dichloro-
5,6-dicyanobenzoquinone (Becker, H. D.; Bjoerk, A.; Adler, E. J. Org.
Chem. 1980, 45, 1596-600) produced the required aldehydes 4.
R′
EtO
HO
O
O
H
O
O
R′
R′
H
O
EtO
O
O
H
OMe
i
OMe
OMe
R
OMe
Ph
ii—v
R
OMe
Ph
R
OMe
O
O
OH
6
4
4gg: R = R′ = H 4gs: R = H, R′ = OMe 4sg: R = OMe, R′ = H 4ss: R = R′ = OMe
Relevant NMR data for samples in acetone-d6 follow; solvent peaks used
as internal reference (1H 2.04, 13C 29.8 ppm). The fully authenticated data
for the four compounds and their acetates will be deposited as compound
numbers 3034-3041 in the “NMR Database of lignin and cell wall model
compounds” available via the Internet at http://www.dfrc.ars.usda.goV/
software.html; Ralph, S. A.; Ralph, J.; Landucci, W. L.; Landucci, L. L.
2000. 4gg (acetate, model for 4GG units): δC/δH (assignment) 188.2/9.55
(9), 149.9/- (8), 135.4/7.30 (7), 133.3/- (1), 114.7/7.68 (2) 124.5/7.45 (6).
4sg (acetate, model for 4SG units): δC/δH (assignment) 188.3/9.57(9),
149.8/- (8), 136.1/7.31(7), 131.4/- (1), 108.1/7.29 (2,6). 4gs (acetate, model
for 4GS units): δC/δH (assignment) 187.0/9.32(9), 152.1/- (8), 126.7/
6.71(7), 133.4/- (1), 115.2/7.68 (2), 124.0/7.54 (6). 4ss (acetate, model for
4SS units): δC/δH (assignment) 187.0/9.32(9), 152.1/- (8), 126.8/6.69 (7),
132.7/- (1), 108.1/7.34 (2,6).
Acknowledgment. We are grateful to USDA-NRI for
partial funding (99-02351 in the Improved Utilization of
Wood and Wood Fiber section), to Fachuang Lu for valuable
discussions, and to Gosta Brunow (University of Helsinki,
Finland) for comments regarding dimerization vs cross-
coupling.
OL005906O
(15) There is some suggestion in spectra from guaiacyl-only lignins and
synthetic lignins (not shown) that coniferyl aldehyde may cross-couple with
other 5-substituted guaiacyl units.
(16) Osakabe, K.; Tsao, C. C.; Li, L.; Popko, J. L.; Umezawa, T.;
Carraway, D. T.; Smeltzer, R. H.; Joshi, C. P.; Chiang, V. L. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 8955-8960. Humphreys, J. M.; Hemm, M. R.;
Chapple, C. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10045-10050.
(13) Sarkanen, K. V. In Lignins, Occurrence, Formation, Structure and
Reactions; Sarkanen, K. V., Ludwig, C. H., Eds.; Wiley-Interscience: New
York, 1971; pp 95-163.
(14) Connors, W. J.; Chen, C.-L.; Pew, J. C. J. Org. Chem. 1970, 35,
1920-1924.
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