Arylpropane-1,3-diols in lignins from normal and CAD-deficient pines

Article abstract of DOI:10.1021/ol9906559

(equation presented) Significant quantities of arylpropane-1,3-diols have been identified in lignins isolated from a CAD-deficient pine mutant; smaller amounts are also present in lignins from normal pine. They arise from dihydroconiferyl alcohol via the action of peroxidases which are responsible for the radical generation steps of lignification. The structures in the complex lignin polymers are proven using 2D and 3D NMR of isolated lignin fractions.

Full text of DOI:10.1021/ol9906559

ORGANIC
LETTERS
1999
Vol. 1, No. 2
323-326
Arylpropane-1,3-diols in Lignins from
Normal and CAD-Deficient Pines
John Ralph,*,†,‡ Hoon Kim,†,‡ Junpeng Peng,† and Fachuang Lu†,‡
U.S. Dairy Forage Research Center, USDA-Agricultural Research SerVice,
Madison, Wisconsin 53706, and Department of Forestry, UniVersity of
WisconsinsMadison, Madison, Wisconsin 53706
jralph@facstaff.wisc.edu
Received May 11, 1999
ABSTRACT
Significant quantities of arylpropane-1,3-diols have been identified in lignins isolated from a CAD-deficient pine mutant; smaller amounts are
also present in lignins from normal pine. They arise from dihydroconiferyl alcohol via the action of peroxidases which are responsible for the
radical generation steps of lignification. The structures in the complex lignin polymers are proven using 2D and 3D NMR of isolated lignin
fractions.
The hydroxyphenylpropanoid polymeric component of plants,
lignin, can be dramatically altered when lignin-biosynthetic-
pathway enzymes are downregulated in mutant or transgenic
plants.1-3 We recently characterized a pine mutant deficient
in cinnamyl alcohol dehydrogenase (CAD),2,4 the enzyme
catalyzing the conversion of 4-hydroxycinnamaldehdes to
4-hydroxycinnamyl alcohols, the monomers principally used
in lignification. During lignification, these monomers are
converted to radicals where they undergo radical coupling
reactions with radicals from other monomers or, more
commonly, from the growing lignin oligomer/polymer to
build up a complex macromolecular structure.5 Because of
the various possible coupling sites on these radicals, the
resulting polymer is complex and contains a variety of
interunit linkages.
Several mutants and transgenics in which the CAD level
is downregulated incorporate the precursor aldehydes of the
normal monolignols into their lignins.6 Thus in a CAD-
deficient tobacco transgenic, sinapaldehyde becomes a major
component of the lignin, radically coupling with other such
aldehyde units and cross-coupling with traditional lignin
components.2 Similarly, in the CAD-deficient mutant pine,
coniferaldehyde units become significant in the polymer.7
But the more striking observation in the pine was the clear
incorporation of significant quantities of an unexpected unit,
dihydroconiferyl alcohol (DHCA), into the lignin.8 DHCA
is found at lower levels in normal plant lignins,9,10 but its
derivation is not completely clear. Despite claims that the
†USDA.
‡ University of Wisconsin.
(1) Ralph, J.; MacKay, J. J.; Hatfield, R. D.; O’Malley, D. M.; Whetten,
R. W.; Sederoff, R. R. Science 1997, 277, 235-239.
(2) Ralph, J.; Hatfield, R. D.; Piquemal, J.; Yahiaoui, N.; Pean, M.;
Lapierre, C.; Boudet, A.-M. Proc. Nat. Acad. Sci. U.S.A. 1998, 95, 12803-
12808.
(3) Boudet, A.-M. Trends Plant Sci. 1998, 3, 67-71.
(4) MacKay, J. J.; O′Malley, D. M.; Presnell, T.; Booker, F. L.; Campbell,
M. M.; Whetten, R. W.; Sederoff, R. R. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 8255-8260.
(5) (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.
(6) Sederoff, R. R.; MacKay, J. J.; Ralph, J.; Hatfield, R. D. Curr. Opin.
Plant Biol. 1999, 2, 145-152, and references therein.
(7) This has now been shown by various methods which act on the whole
cell wall material,6,9 not just the isolated lignin examined by NMR methods.1
10.1021/ol9906559 CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/22/1999

unit can only arise as a modified metabolic product following
dimerization of traditional lignin monomers,11 we have
presented ample evidence that it is formed as a monomer
and that the monomer is incorporated into the lignin via
radical coupling processes.1,6,9 Additionally, DHCA monomer
is found in solvent extracts from these plants.6 It is however
still not known whether it derives from coniferyl alcohol or
can come via other pathways from coniferaldehyde. Since
all indications in the pine mutant are that coniferaldehyde
builds up and is not efficiently reduced to the alcohol, the
significant DHCA component was conjectured to arise from
coniferaldehyde rather than coniferyl alcohol,1 but this has
not yet been elucidated.
Here we identify another significant component of the
mutant pine’s lignin, arylpropane-1,3-diols, whose source
was originally puzzling. It is now clear that these units derive
directly from DHCA by peroxidase-mediated reactions.
Figure 1a shows a subplot of the side chain region from
a 2D HMQC-TOCSY NMR experiment on acetylated lignin
isolated from the mutant pine,12 highlighting the new
arylpropane-1,3-diacetates (red) along with the previously
identified DHCA units (green). Data from a model com-
pound, 1,3-diacetoxy-1-(4-O-benzyl-3-methoxyphenyl)pro-
pane, 5-Ac,13 are at the center of the yellow circles and
obviously match well.
correlations from other units with protons resonating in the
same regions. Thus, resonances at 2.2 ppm arise only from
the new unit (â-proton) and DHCA (â-proton) (as well as
the strong acetone solvent signal). Figure 1e is therefore a
composite HSQC of those units, namely, the acetone, as well
as DHCA (green) and APD (red). The plane through HR (5.8
ppm, Figure 1d) is quite clean but the TOCSY transfer
between HR and Hâ is poor (under the chosen acquisition
conditions), giving rise to only weak Hâ/Câ and Hγ/Cγ cross-
peaks. γ-Protons in lignin seriously overlap; consequently
the plane through an Hγ (4.1 ppm, Figure 1f) shows the nice
correlations for the APD unit, but also with DHCA (green),
lignin’s â-aryl ether units (blue), and the intense methoxyl.
The observation of complete HSQC spectra for the ADP unit
in all three planes corresponding to the APD side chain
protons (Figures 1d-f), along with the evidence from the
2D-HMQC-TOCSY (Figure 1a), provides sufficiently com-
pelling proof of the structure in the isolated lignin from the
CAD-deficient pine mutant. A model compound for etheri-
fied APD structures, 5,13 provides data for comparison,
Figure 1g; note however that there are a large number of
different bonding environments in lignin so the peaks are
broader and more disperse in lignin spectra.9
As with other novel units found in lignins from transgenic
or mutant plants, traces of the same components can be found
in lignins from control plants. Figure 1b shows the R-C/H
region of HSQC spectra of the mutant’s lignin, where the
â-aryl ether units (blue) and the strong new ADP unit (red)
appear most cleanly. Figure 1c shows the same region in
lignin from a normal pine control. Although the peak is weak,
it is diagnostic and, with the other correlations evident (not
shown), well authenticated. The peak is also in lignins
isolated from mature pine clear sapwood.
Perhaps more diagnostic is the data from a 3D TOCSY-
HSQC experiment.14 2D F2-F3 planes from this experiment
show a complete HSQC spectrum of any units bearing
protons resonating at the frequency of the taken slice.9 In
spectra as complex as those from lignins, finding unique
resonances in the proton spectrum is often difficult. However,
slices through each of the three side chain protons in APD
units show rather clear HSQC spectra of that unit, along with
Where do APD units come from? They derive from DHCA
2 via the action of peroxidase and hydrogen peroxide,
Scheme 1. The mechanism, via a vinylogous quinone
(8) Lignin is the term used for the phenylpropanoid polymer in the cell
walls of normal plants and will be used here although there remain issues
regarding the biochemical and functional roles of “nontraditional” lignins
in mutants and transgenics (see refs 6 and 11).
(9) Ralph, J.; Marita, J. M.; Ralph, R. A.; Hatfield, R. D.; Lu, F.; Ede,
R. M.; Peng, J.; Quideau, S.; Helm, R. F.; Grabber, J. H.; Kim, H.; 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; in press.
Scheme 1
(10) (a) Lundquist, K.; Stern, K. Nordic Pulp Paper Res. J. 1989, 210-
213. (b) Fukagawa, N.; Meshitsuka, G.; Ishizu, A. J. Wood Chem. Technol.
1991, 11, 373-396. (c) Brunow, G.; A¨ mmålahti, E.; Niemi, T.; Sipila¨, J.;
Simola, L. K.; Kilpela¨inen, I. Phytochem. 1998, 47, 1495-1500.
(11) (a) Gang, D. R.; Fujita, M.; Davin, L. D.; Lewis, N. G. In Lignin
and Lignan Biosynthesis; Lewis, N. G., Sarkanen, S., Eds.; American
Chemical Society: Washington, DC, 1998; ACS Symposium Series 697,
pp 389-421. (b) Lewis, N. G.; Davin, L. B.; Sarkanen, S., ref 11a, pp
1-27. (c) Lewis, N. G. In Abstracts of Papers, 215th National Meeting of
the American Chemical Society, Dallas, TX; American Chemical Society:
Washington, DC, 1998; Vol. 1, p Cell-09.
(12) NMR spectra were taken on a Bruker DRX-360 instrument fitted
1
with a 5 mm H/broadband gradient probe with inverse geometry (proton
coils closest to the sample). The conditions used for all samples were ∼80
mg of acetylated isolated lignin in 0.4 mL of acetone-d6, with the central
solvent peak as internal reference (δH 2.04, δC 29.80). Experiments used
were standard Bruker implementations of inverse (1H-detected) 2D-gradient-
HSQC, 2D-HMQC-TOCSY, and 3D-gradient-TOCSY-HSQC experiments.
The TOCSY spin lock period was 100 ms. Carbon/proton designations are
based on conventional lignin numbering. Lignin 2D and 3D spectra have
been extensively detailed in a recent book chapter.9 The CAD-deficient
pine mutant and the isolation of lignin fractions have been described
previously.1 For this study, the lignins were acetylated, extracted into EtOAc,
and washed with 6 mM EDTA to improve NMR relaxation properties (cf.
ref 2).
methide 4, involves two H-radical abstractions. Abstraction
from a benzylic CH2 to produce quinone methides from
phenoxy radicals has been noted previously.15 When DHCA
is subjected to peroxidase-H2O2, monomeric APD 1 as well
as the range of homo dimers and crossed dimers 7-11
(Scheme 2) involving DHCA and APD are found, as will
be detailed elsewhere.
324
Org. Lett., Vol. 1, No. 2, 1999

Figure 1. NMR spectra12 showing new APD structures (red) and derived benzylic ketone analogues (magenta), along with DHCA units
(green); some â-aryl ether units are also shown (blue); data from acetylated model 5 are in yellow: a-c and g are from 2D experiments;
d-f are 2D planes from a gradient-selected 3D-TOCSY-HSQC experiment.
Org. Lett., Vol. 1, No. 2, 1999
325

the ball-milling step). Ketones 6 provide additional confir-
matory evidence for the APD structures 1 described above.
The finding of significant quantities of APD units in the
mutant’s lignin is further evidence for the involvement of
DHCA monomers in this plant’s lignification. Nevertheless,
some APD units likely arise following radical homo-coupling
of DHCA 2 to give 5-5- or 4-O-5-coupled units 7 and 8,
Scheme 2; each of these units retains a free-phenolic DHCA
moiety D which can be hydroxylated to give units A,
structures 9-11, by the same mechanism as shown in
Scheme 1. As additional proof, degradation of the mutant’s
lignin by “derivatization followed by reductive cleavage” (the
DFRC method)17 produces the monomer APD 1 as well as
numerous 5-5- and 4-O-5- products of homo- and cross-
coupling (manuscript in preparation).
Scheme 2
In conclusion, another previously unidentified unit present
in small quantities in normal lignins is a major component
of the hydroxyphenylpropanoid polymeric component of a
pine mutant deficient in CAD. Those units, arylpropane-1,3-
diols, arise from dihydroconiferyl alcohol monomers by
radical reactions. Lignin-biosynthetic-pathway mutants and
transgenics provide a rich source of insight into details of
the chemistry and biochemistry of hydroxyphenylpropanoid
polymer formation.
Also evident in the HMQC-TOCSY spectrum, Figure 1a,
are (acetylated) ketones 616 (magenta contours). Products of
benzylic alcohol oxidation are seen in various isolated lignins,
notably from syringyl (3,5-dimethoxy-4-hydroxyphenyl)
units;9 they may arise during lignin isolation (particularly in
Acknowledgment. We thank John J. MacKay (Institute
of Paper Science and Technology, Atlanta, GA) and Ronald
R. Sederoff (Department of Genetics, North Carolina State
University, Raleigh, NC) for providing the mutant pine that
was used for this work and the original study described in
ref 1, the U.S. Dairy Forage Research Center for funding
NMR upgrades that made the gradient experiments possible,
and partial funding through USDA-NRI competitive grants
96-35304-3864 (Plant Growth and Development) and 97-
02208 (Improved Utilization of Wood and Wood Fiber).
(13) Preparation of APD model 1,3-dihydroxy-1-(4-O-benzyl-3-meth-
oxyphenyl)propane 5:
(i) BnCl, Me2CO, K2CO3; (ii) BrCH2COOEt, Zn, NH4Cl, THF (Tanaka,
K.; Kishigami, S.; Toda, F. J. Org. chem. 1991, 56, 4333-4334); (iii)
DIBAL, toluene. Diacetate of compound 5, δC/δH: 73.2/5.85 (R), 35.8/
2.18 (â), 61.2/4.08 (γ). Unambiguously assigned full NMR data will be
deposited in the Internet-accessible NMR Database of Lignin and Cell Wall
Model Compounds (Ralph, S. A.; Ralph, J.; Landucci, W. L.; Landucci, L.
L.; http://www.dfrc.ars.usda.gov/software.html 1999, entry #3029) Various
APD-containing products have also been isolated from radical coupling
reactions of DHCA, as will be described elsewhere.
(14) (a) Schleucher, J.; Schwendinger, M.; Sattler, M.; Schmidt, P.;
Schedletzky, O.; Glaser, S. J.; Sorensen, O. W.; Griesinger, C. J. Biomol.
NMR 1994, 4, 301-306. (b) Palmer, A. G.; Cavanagh, J.; Wright, P. E.;
Rance, M. J. Magn. Reson. Ser. A 1991, 93, 151-170.
(15) Zanarotti, A. Tetrahedron Lett. 1982, 23, 3815-18.
(16) NMR data for units 6-Ac, δH/δC (assignment): 3.31/37.5 (â), 4.41/
60.4 (γ), 196.3 (R).
Supporting Information Available: Full NMR data for
diacetate of 5. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL9906559
(17) (a) Lu, F.; Ralph, J. J. Agric. Food Chem. 1997, 45, 2590-2592.
(b) Lu, F.; Ralph, J. J. Agric. Food Chem. 1997, 45, 4655-4660. (c) Lu,
F.; Ralph, J. In Lignin and Lignan Biosynthesis; Lewis, N. G., Sarkanen,
S., Eds.; American Chemical Society: Washington, DC, 1998; pp 294-
322
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