Stereoselective phenolic coupling in Blechnum spicant: Formation of 8-2′ linked (-)-cis-blechnic, (-)-trans-blechnic and (-)-brainic acids

Article abstract of DOI:10.1039/b008174o

In vivo administration experiments using stable (¹³C) and radio (¹⁴C) labeled precursors provide further evidence for vascular plant proteins engendering specific but distinct phenolic coupling modes, i.e. in this case for stereosele

Full text of DOI:10.1039/b008174o

Stereoselective phenolic coupling in Blechnum spicant: formation of 8–2A linked
(2)-cis-blechnic, (2)-trans-blechnic and (2)-brainic acids†
Chang-Zeng Wang, Laurence B. Davin and Norman G. Lewis*
Institute of Biological Chemistry, Washington State University, Pullman, USA WA 99164-6340.
E-mail: lewisn@wsu.edu; Tel.: 509-335-2682; Fax: 509-335-8206
Received (in Corvallis, OR) 9th October 2000, Accepted 14th November 2000
First published as an Advance Article on the web
In vivo administration experiments using stable (13C) and
radio (14C) labeled precursors provide further evidence for
vascular plant proteins engendering specific but distinct
phenolic coupling modes, i.e. in this case for stereoselective
8–2A coupling leading to the optically active lignans,
(–)-blechnic and (–)-brainic acids.
5 was also isolated, which on standing in open solution (in
CH3CN or H2O) over 72 h, was converted into (2)-trans-
blechnic acid 1. Compound 5 ([a]D 2128°, c 0.58, MeOH) had
a molecular formula of C18H14O8 as evidenced from its HR-MS
1
(381.7373 [M+Na]+); furthermore, comparison of its H and
13C NMR spectral data with those of (2)-blechnic acid (2)-1
revealed that both structures were very similar, except for a cis-
double bond in 5 [1H NMR: 6.74 (1H, d, J = 12.7 Hz, H-7A),
5.88 (1H, d, J = 12.7 Hz, H-8A); 13C NMR: 139.7 (C-7A) and
120.2 (C-8A)] vs. the trans double bond in blechnic acid 1 [1H
NMR: 7.57 (1H, d, J = 16.1 Hz, H-7A), 6.27 (1H, d, J = 16.1
Hz, H-8A); 13C NMR: 143.3 (H-7A) and 117.8 (H-8A)]. Correla-
tions (or connectivities) for compound 5 were determined by
2D-NMR (1H-1H COSY, HMQC, HMBC) spectroscopic
analyses conducted at low temperature (225°C), and the
configuration of the cis-double bond was further confirmed by
both NOE experiments and a comparison to that of (2)-trans-
blechnic acid 1.
The ferns (Pteridophytes) are evolutionary forerunners of the
gymnosperms and angiosperms. They contain various phenolic
natural products which may represent some of the earliest
examples in planta of control of stereoselective phenolic
coupling. For example, Blechnum spicant (deer fern) and B.
orientale1 accumulate the unusually linked, optically active,
8–2A lignans, (2)-blechnic 1 and (2)-brainic 2 acids.
The precursor relationships to the optically active 8–2A linked
lignans was examined through deployment of radiolabeled Phe
6, Tyr 7, cinnamic 8, p-coumaric 9 and acetic acids as potential
precursors over extended durations (Table 1). Thus, caffeic acid
3, 5-caffeoyl shikimate 4, (2)-cis-blechnic 5, (2)-trans-
blechnic 1 and (2)-brainic 2 acids, were individually isolated
with the relative total radiochemical incorporation for each
estimated by liquid scintillation counting (see Table 1).
Significantly, a rapid incorporation into (2)-cis-blechnic acid 5
( ~ 3%) was noted within 8 h, relative to that of (2)-trans-
blechnic 1 and (2)-brainic 2 acids (@0.1%). This indicated that
(2)-cis-blechnic acid 5 was the initial coupling product, since it
was apparently further metabolized into (2)-trans-blechnic 1
and (2)-brainic 2 acids based on the trends of total incorpora-
tion noted for 1 and 2 which approached c. 1–2 and 3–4% over
48–84 h, respectively. A less likely interpretation is that 5 might
be a shunt metabolite and not directly involved in the formation
of 1 and 2. For each time frame examined, the relative
incorporation into (2)-brainic acid 2 was higher than that into
(2)-blechnic acid 1, suggesting that cis-blechnic acid 5 was
more effectively channeled into (2)-brainic acid 2 rather than
into (2)-blechnic acid 1. In a somewhat analogous manner,
both [3-14C]cinnamic 8, and [2-14C]p-coumaric 9 acids served
Gymnosperm and angiosperm lignans are frequently derived
from stereoselective 8–8A coupling of two monolignol
(hydroxycinnamyl alcohol) radicals,2 and the recent discovery
of dirigent proteins (Latin: dirigere, to guide or to align) and
their corresponding genes gave a new perspective into how free
radical coupling is controlled in plants in vivo.3–5 There have
been no reports on the stereoselective control of other coupling
modes, and hence establishing the mechanism of 8–2A linked
lignan formation in the Blechnaceae would be instructive.
The MeOH extract of B. spicant contains as its principal
metabolites, caffeic acid 3, 5-caffeoyl shikimate 4, (2)-trans-
blechnic acid 1 and (2)-brainic acid 2. An unknown compound
as precursors, whereas neither [U-14C]tyrosine
7 nor
[2-14C]NaOAc were incorporated into any of the various
metabolites of B. spicant examined, (data not shown).
L
-[3-13C], [2-13C] and [1-13C] phenylalanine 6 (1.0 mM)
were next individually administered to B. spicant fronds for 5
days, with the resulting (2)-brainic 2 and (2)-trans-blechnic 1
acids isolated by preparative mBondapak C18 column HPLC
and subjected to 13C-NMR spectroscopic analyses (see Fig. 1a–
d). Only the data for (2)-brainic acid 2 are presented. Fig. 1a
shows the natural abundance NMR spectrum and the corre-
sponding assignments for (2)-brainic 2 acid; these are based on
† Electronic supplementary information (ESI) available: 13C-NMR spectra
of (a) natural abundance (2)-trans-blechnic 1 acid, as well as (2)-trans-
blechnic acids 1 obtained following administration of (b) [1-13C], (c)
[2-13C] and (d) 3-13C]-phenylalanine 6 (1 mM) to B. spicant fronds for 5
days; all spectra were recorded under identical conditions.
The relative carbon-13 enrichments noted were higher for (2)-brainic
acid 2 than for (2)-trans-blechnic 1 due in large part to the much higher
endogeneous levels of the latter in B. spicant. See http://www.rsc.org/
suppdata/cc/b0/b008174o/
1
2-D NMR spectroscopic analyses (HMQC, HMBC and H-1H
COSY) and previous assignments by Wada et al.1 The
(2)-brainic 2 acid obtained following administration of
[1-13C]Phe 6 displayed carbon-13 enriched resonances for C-9
and C-9A at 170.5 and 170.6 ppm, respectively (Fig. 1b). In a
DOI: 10.1039/b008174o
Chem. Commun., 2001, 113–114
This journal is © The Royal Society of Chemistry 2001
113

Table 1 Incorporation of 14C-labeled potential precursors into various B. spicant phenolic metabolites (% incorporation)
Caffeic
acid 3
(% incor-
poration)
(2)-
(2)-cis-
trans-
Blechnic
acid 1
(2)-
Brainic
acid 2
Metabolic
period (h)
5-Caffeoyl
Blechnic
Potential precursor administered (radio-activity)
shikimate 4 acid 5
A
[U-14C] Phenylalanine 6 (200 mL, 148 kBq, 18.5 GBq nmol21
)
4
8
1.2
2.3
2.3
2.0
2.9
2.9
5.0
4.5
2.8
2.9
3.8
2.7
1.0
1.7
3.4
2.5
3.2
4.1
5.1
4.7
5.3
3.3
6.9
5.6
5.6
3.0
1.1
3.0
2.6
3.9
2.3
2.7
2.0
1.4
1.3
3.5
1.0
1.2
1.4
< 0.1
< 0.1
0.1
0.5
0.6
0.7
0.7
1.1
2.1
2.0
1.1
1.5
0.5
< 0.1
0.1
0.4
1.4
1.6
1.6
1.9
2.9
4.2
1.5
1.4
1.2
0.7
12
18
24
30
36
48
84
24
50
84
24
B
C
[3-14C] Cinnamic acid 8 (200 mL, 185 kBq, 1.89 GBq nmol21
)
[2-14C] p-Coumaric acid 9 (200 mL, 148 kBq, 6.07 GBq nmol21
)
Scheme 1 Proposed stereoselective coupling of caffeic acid 3 leading to
(2)-trans-blechnic acid 1.
trans-caffeic acid 3 to first afford (2)-cis-blechnic acid 5. Less
likely, stereoselective coupling could involve p-coumaric acid 9
with subsequent hydroxylation of the aromatic ring to ulti-
mately afford the corresponding catechols 1 and 2. In any case,
by comparison to the dirigent mediated stereoselective bimo-
lecular coupling of coniferyl alcohol 10 affording (+)-pinor-
esinol 11, it is tempting to suggest that the formation of the
blechnates 1 and 2 may involve a comparable process.4,5 This
will be established in the future by isolation and character-
ization of the relevant proteins and enzymes involved in the
biosynthesis of (2)-trans-blechnic acid 1 and its derivatives in
B. spicant; nevertheless both 8–8A and 8–2A linked phenyl-
propanoid coupling products are under full proteinaceous
control thereby stipulating the outcome of stereoselective
coupling.
The authors thank the USDOE (DE-FG03-97ER20259) and
the NSF (MCB-9976684), as well as the L. B. and D. Cullman
and G. T. Hargrove Center for Land Plant Adaptation.
Fig. 1 13C-NMR spectra of (a) natural abundance (2)-brainic 2 acid, as well
as (2)-brainic acids 2 obtained following administration of (b) [1-13C], (c)
[2-13C] and (d) [3-13C]phenylalanine 6 (1 mM) to B. spicant fronds for 5
days; all spectra were recorded under identical conditions.
Notes and references
1 H. Wada, T. Kido, N. Tanaka, T. Murakami, Y. Saiki and C. M. Chen,
Chem. Pharm. Bull., 1992, 40, 2099.
2 N. G. Lewis and L. B. Davin, Lignans: Biosynthesis and Function, in
Comprehensive Natural Products Chemistry, Vol. 1, ed. Sir D. H. R.
Barton, K. Nakanishi and O. Meth-Cohn, Elsevier, London, pp
639-712.
similar manner, administration of [2-13C]Phe 6 gave enhanced
signals for the C-8 and C8A resonances of (2)-brainic 2 acid at
55.4 and 118.2 ppm (Fig. 1c), whereas with [3-13C]Phe 6, the
corresponding enhanced signals at C-7 and C7A were at 88.0 and
142.7 ppm (Fig. 1d). Comparable results were observed for
(2)-trans-blechnic acid 1 biosynthesis (see ESI†).
3 L. B. Davin and N. G. Lewis, An. Acad. Bras. Cienc. Suppl. 3, 1995, 67,
363.
We propose that the 8–2A linked lignans, (2)-blechnic 1 and
(2)-brainic 2 acids, result from oxidative coupling of caffeic
acid 3 as depicted in Scheme 1. Based on the trends of relative
incorporations into 5, 1 and 2, this conversion is likely to result
via stereoselective coupling of two molecules of either cis- or
4 L. B. Davin, H. B. Wang, A. L. Crowell, D. L. Bedgar, D. M. Martin, S.
Sarkanen and N. G. Lewis, Science, 1997, 275, 362.
5 D. R. Gang, M. A. Costa, M. Fujita, A. T. Dinkova-Kostova, H. B. Wang,
V. Burlat, W. Martin, S. Sarkanen, L. B. Davin and N. G. Lewis, Chem.
Biol., 1999, 6, 143.
114
Chem. Commun., 2001, 113–114