Phytochemwy, Vol 29, No 6, pp 1841-1846, 1990

Prmted I Great Brltam

003 l-9422/90 $3 00 + 0 00
0 1990 Pergamon Press plc
BIOSYNTHESIS OF LIGNANS IN FORSYTHIA INTERMEDIA
MAIADA M A. RAHMAN, PAUL M. DEWICK, DAVID E. JACKSON and JOHN A. LUCAS
Departments of Pharmaceuttcal Sciences and Botany, Umversrty of Nottmgham, Nottmgham NG7 2RD, U K
(Recerued 18 September 1989)
Key Word Index--Forsythta mtermedza, Oleaceae, hgnans, dtbenzylbutyrolactone, furofuran, blosynthests.
Abstract-Feeding experiments with young shoots of Forsythia rntermedza have shown that phenylalanme and ferulic
acid are good precursors of the dibenzylbutyrolactone hgnan arctigenin and the furofuran lignans phillygenin and
eptpmoresinol. Although matanesmol was mcorporated into arcttgenin, and eptpinoresinol into phillygenin, in accord
with late methylation steps m the btosynthettc pathway, some incorporation of label from one lignan class into the
other was observed, parttcularly from eptpmoresmol mto arctigenin. This may indicate a high degree of catabolic
activny followed by non-specific mcorporation [2-14C,1-3H]Coniferyl alcohol was incorporated into all three
Iignans with a significant increase m the H: 14C isotopic ratto This supports the hypotheses that the hgnans arise by
oxtdattve coupling of two comferyl alcohol units, rather than from two ferulic acid or coniferaldehyde units. The
observed increase in isotopic ratio probably results from a primary tsotope effect in the reverstble oxidation-reduction
reactions interrelating cmnamtc acids and cmnamyl alcohols. A similar mcrease m tsotoptc ratio was measured during
biosynthesis of the aryltetrahn lactone lignan podophyllotoxin m Podophyllum hexandrum.
INTRODUCTION
Lignans are generally believed to be formed by a phenohc
oxidative coupling process analogous to that proposed
for the btosynthesis of the plant polymer hgnin [l, 21.
Monolignol monomers, such as coniferyl alcohol (4), are
postulated to be dehydrogenated to phenoxy radicals;
coupling of these radicals, or mesomeric forms of them,
leads to intermediate qumone methide structures. Dilig-
nols (lignans and neolignans) then arise by attack of
suitable nucleophiles onto the quinone methides, fol-
lowed perhaps by further cyclization and modificatton
processes. Because the majority of natural hgnans are
produced m optically active form, the biosynthettc reac-
tions are suggested to be enzyme-catalysed, rather than
random free radical couplings which may occur during
lignin biosynthesis. There seems to be no conclusive
evidence on whether lignans and neolignans are inter-
mediates in lignin biosynthesis, or whether they represent
a competitive pathway for utilization of lignin precursors.
These proposals are generally borne out by the experi-
mental evidence available, but lignan biosynthesis is a
largely unexplored area, many crucial questions remain
unanswered, and the range of structural types for which
information has been obtained 1s limited [3]. The lignan
profile of Forsythia intermedia [4] afforded us the oppor-
tunity to investigate the biosynthetic ortgms of hgnans
from two different classes, furofuran dertvattves and
dibenzylbutyrolactone derivatives. The latter class of
lignans had been implicated as important intermediates
in the pathway to the more complex aryltetralin lactone
lignans of Podophyllum [S] Although the later stages of
the pathway to the Podophyllum lignans are now fairly
well characterized [S-7], stages prior to dibenzylbu-
tyrolactone intermediates are ill-defined.
Stijcktgt and Klischies have earlier studied the biosyn-
thesis of Forsythia lignans using shoots of F suspensa var
fortunei [8]. In a series of double-labelling experiments,
they demonstrated intact incorporation of ferulic acid (l),
coniferyl alcohol (4) and coniferaldehyde (3), fed as their
glucose derivatives, into arctiin (10) and phillyrm (14).
Glucoferulic acid proved to be the best precursor for both
arctiin and phillyrin. 3,4-Dimethoxycinnamic acid was
not incorporated, in keeping wtth a phenohc oxidative
coupling mechanism. These authors also briefly reported
that m feeding experiments with double-labelled
[14C3H,0H]coniferin, there was no reoxidation to feru-
lit acid and subsequent dimerization at that oxidation
level, inferring that coupling occurs between coniferyl
alcohol units. Fujimoto and Higuchi had also reported
mcorporations of phenylalanine, ferulic acid and sinapyl
alcohol mto the furofuran lignan syringaresinol (15) and
tts diglucoside liriodendrm (16) in Liriodendron tulipifera
[9]. In contrast, coniferyl alcohol and sinaptc acid were
poorly utihzed and coupling of sinapyl alcohol units was
deduced.
The present commumcation describes feeding experi-
ments to investigate m more detail the biosynthetic
pathways to the furofuran derivatives epipmoresinol(11)
and phillygemn (13), and to the dtbenzylbutyrolactone
derivative arctigenin (9) in F. intermedia. These com-
pounds are present both as free lignans and as the
glucosides epipinoresinol 4-O-glucoside (12), phillygemn
4-0-glucostde (phtllyrm, 14), and arcttgenm 4-O-
glucostde (arctiin, 10) [4] Although matatresinol(7) and
its 4-0-glucoside (8) are also present m low concen-
trations, the quantittes dtd not permit biosynthetic study
of these compounds.
RESULTS AND DISCUSSION
Feeding experiments
Earlier feeding experiments using F. suspensa had
employed young shoots and precursors were admin-
1841
1842 M M A RAHMAN et al
Table 1 IncorporatIon data from feedmg of oL-[l-4C]phenylalamne to Forsythra zntermedra shoots
Feedmg Arctlgenm Phillygemn Eplpmoresmol
time (hr) Expt mg % Incorp Dilution mg % Incorp Dilution mg % Incorp Dilution
24 (1) 124 3 56 278 2 64 030 695 6 50 0 30 1598
(10 162 3 52 368 2.18 031 545 8 45 0 32 1947
48 (1) 99 2 83 277 2 10 040 409 200 0 38 384
(11) 106 321 264 2 35 042 461 2 50 041 448
72 (1) 104 283 292 2.43 042 478 4 64 051 677
(11) 77 2 63 233 1 63 041 321 3 27 0 50 494
Table 2 Incorporation of [3-O-4CH,]ferullc acid into For-
sythza zntermedza hgnans
Lignan
mg
% Incorporation Ddution
Expt (1)
Arctigemn
Phlllygenm
Eplpmoresmol
Expt (n)
Arctigenm
Phlllygenm
Eplpmoresmol
489 190 112
099 033 131
4 38 1 35 132
4 76 244 85
081 031 111
3 52 1 36 105
istered in aqueous solution through the stem [8]. This
resulted in excellent incorporations; a similar technique
was thus tested and used m the present studies. Shoot tips
some 4-7 cm long were excised from the parent shrub
during the active growing season and dipped into solu-
tions of the radloactive precursors (ca 1 mg), solubilized
in water, NaOH, or DMSO-NaOH as appropriate.
Distilled water was added as necessary during the feeding
period. After metabohsm, the.plant tissue was homogen-
ized and extracted with methanol The crude extract was
then hydrolysed with P-D-glucosldase to liberate lignan
aglycones from their glucosldes, so that durmg work-up,
aglycones and glucosldes were thus assayed together.
Partial purification of the hgnans was obtained by par-
tition between CH,Cl,-water, then by prep. TLC of the
organic-soluble fraction.
Arctlgenm and phillygenm cochromatographed on
TLC [4]. and were thus acetylated (Ac,O-pyridine) prior
to separation by TLC. Epipmoresmol was slmllarly de-
rivatlzed by acetylatlon The three acetates were quantl-
fied by UV absorption, then rigorously purified by
chromatography and/or repeated recrystallization to
constant specific activity, prior to radiochemlcal count-
mg. A single shoot normally yielded sufficient lignans for
purification without ddutlon Duplicate experiments
were conducted.
Preliminary mcorporatlon experiments with DL-[ l-
C]phenylalanme (Table 1) demonstrated a high level of
biosynthetic actlvlty in the shoots. After three different
feeding periods, 24,48 and 72 hr, good incorporation mto
all three lignans was recorded, with very high mcorpora-
tions of 2 6-3.5% being observed for arctigenin. The
highest mcorporatlon into arctlgenm was noted after
24 hr feeding, with a maximum for phillygenin m the
48-72 hr feedmg period with eplpinoresmol appearing to
peak later. However, variations m mcorporatlon at the
different times was not great and all feedings gave very
satisfactory mcorporatlon. [3-014CH,]Ferulic acid was
subsequently fed to shoots for a period of 72 hr, most of
this time being necessary for absorption of the precursor
solution High mcorporatlons mto all three lignans, partl-
cularly into arctigemn and eplpmoresmol were recorded
(Table 2)
Based on structural analysis of the hgnans m F. znter-
medza [4], a biosynthetic sequence may logically be
postulated as in Scheme 1. The two groups are envisaged
to arise by alternative reactions on a common quinone
methlde intermediate (6), derived by couphng of free
radicals (5), orlgmatmg from two units with the feruhc
substitution pattern, here presumed to be comferyl al-
cohol (4) The furofuran lignans are produced from this
qumone methide by stereospecific nucleophdic addltlons,
whilst the dlbenzylbutyrolactones require reduction and
formation of the lactone ring. Methylatlon reactions are
presumed to be late m the sequence, based on data from
cell cultures where certain pathways are blocked or
inhibited [lo], and the non-mcorporatlon of 3,4-dimeth-
oxycmnamic acid mto the lignans of F. suspensa [S]
Glucosylation reactions are slmdarly predicted to be late
processes since fresh tissue contams both glucosldes and
aglycones. If this scheme does operate, then feeding
experiments with matalresmol(7) and eplpinoresmol(l1)
should give evidence for the methylation reactions.
When fed to shoots of F. zntermedra, [ 14C] matalresmol
proved to be an excellent precursor of arctlgemn (m-
corporations of 0.50 and 1 11%) (Table 3), as anticipated
if 4-O-methylatlon 1s a late step. Small, but not totally
insignificant incorporations mto epipinoresmol and phil-
lygenin were also recorded. The complementary feeding
of [14C]eplpmoresinol was anticipated to label philly-
genm via methylatlon and this was observed (incorpor-
ations of 0.23 and 0.24%). However, in these experiments,
arctlgenin also became labelled (mcorporatlon of 0.83%).
The low percentage mcorporatlon values mto phdlygemn
probably reflect the lower abundance of this lignan m the
plant, but the arctlgemn figures suggest some other,
unexpected metabolism. From the combined results,
there exists the possibility that eplpinoresmol, and to a
lesser extent matairesinol, is capable of being transformed
back to the qumone methlde (6) and of then being
mcorporated into the other class of lignan. Alternatively,
and probably more likely, a high degree of catabohc
activity exists m Forsythru and added hgnans are being
degraded and incorporated non-speclfically. This is borne
out by the observed rapid catabolism of hgnans in cell
cultures of F. tntermedza [lo] If this is the case, then even
the observed methylation of matairesinol and
Lignan biosynthesis m Forsythm
1843
8 6
ll
OGlC OH
7
6
Scheme 1 Proposed biosynthetic pathway to hgnans m Forsythra intermedw
Table 3 Incorporation of [*CjmatairesmoI and [SCJepipmoresmol mto Forsythia rntermedm hgnans
Lignan fed
Arctigenm Phillygemn Eptpmoresmol
Expt mg % Incorp Dilution mg % Incorp Dtlution mg % Incorp Dilution
Matairesmol (I) 8.42 111 426 2.09 0.017 6740 5.95 0072 4330
(11) 668 0 50 753 1.52 0.011 7920 2.87 0.090 1680
Epipmoresmol (I) 6.13 0.83 800 1.35 023 629
01) 6.80 0.83 1067 193 0.24 1034
epipinoresinol may be the result of non-specific processes substitution patterns on the aromatic rings at the coup-
and further study is required. ling stage, we had acquired little data on the oxidation
Our experiments on lignan biosynthesis using phenyl- level of the side-chain at coupling. By analogy with lignin,
propanoid precursors had employed mainly cinnamic coupling of cinnamyl alcohols is widely assumed, though
acids [ 111. Although these gave useful information about definitive evidence is sparse. Accordingly, to follow reten-
1844 M. M A RAHMAN et al
tlon of hydrogen from the primary alcohol function of
coniferyl alcohol on incorporation into various classes of
hgnans, [2-4C,1-3H]conlferyl alcohol was fed to F.
rntermedia shoots, and also to roots of Podophyllum hexand-
rum, the latter plant producmg the aryltetralm lactone
podophyllotoxm (17) Depending on whether two comferyl
alcohol, ferulic acid, or coniferaldehyde units couple, or
combmatlons of these, it IS possible to predict the level of
t&urn retention that might result in each type of lignan.
The results are presented in Table 4. In all cases, however,
the labelled hgnans isolated from the feeding experiments
showed a significant increase in the 3H. 14C ratlo over
that of the precursor fed, even when at least half of the 3H
label should have been lost m, for example, arctigenin and
podophyllotoxm, due to formation of the lactone
functions.
In view of the observed mcorporatlon of label from one
class of Forsythia hgnan mto the other, the previously
reported [ 1 l] degradation and reincorporation mto
podophyllotoxin of label from 3,4-methylenedioxycm-
namic acid, and the unusual degradation of phenylpro-
pane precursors during brosynthesis of allylbenzenes
[12-151, partial or complete degradation of the comferyl
alcohol precursor was considered to be operative here,
and the reason for the unexpected results m Table 4.
However, partial degradation [5, 1 l] of the podophyllo-
toxm sample (3H: 14C= 12.5) to the trlacetate (18)
(3H 14C = 12.5), removing two methyl groups from the
pendent rmg, was accompanied by no loss of 3H label
relative to 14C. This seems to preclude the posslbihty of
trltmm label from the comferyl alcohol entering the C,
pool and subsequently bemg remcorporated mto the
lignan. The most likely explanation for the change m
Isotope ratio on gomg from comferyl alcohol to the
lignans is the known reversibility of enzymlc reactions
interrelating cmnamlc acids and cinnamyl alcohols
[16]. Both cmnamoyl-CoA reductase [cmnamoyl-
CoA NADP oxidoreductase] and cinnamyl alcohol de-
hydrogenase [cinnamyl alcohol.NADP oxldoreductase]
catalyse reversible reductions and together are mvolved
in transforming cmnamoyl-CoA esters to the correspond-
ing cinnamyl alcohols, each requiring NADPH as
cofactor. The consequence of this is that labelled comferyl
alcohol may be oxidized and thus lose some, or all of Its
hydrogen from the primary alcohol function. The
coniferyl alcohol fed was a mixture of two single-labelled
speaes, [2-4C]- and Cl-Hlconiferyl alcohols, and
oxidation will be subject to a primary isotope effect in the
case of the latter specres, though not for the former This
will result m the comferyl alcohol pool gradually becom-
mg more enriched with 3H relative to 14C, and giving the
observed increase in isotope ratio. Perhaps the trltiated
NADPH formed as a by-product during the enzymic
oxidation will also be available to enhance the ratio even
further by tntmm-labellmg of endogenous inactive cin-
namic precursors. In both Forsythia experiments,
eplpmoresmol isolated was found to have a higher
3H. 14C isotope ratio than erther arctlgemn or phllly-
gemn. This 1s most probably a kmetlc effect resulting from
the rate of coniferyl alcohol metabohsm and Its rem-
corporation into the various hgnans
Slmllar feeding experiments with [4C3H,0H]-
comferm m F suspensa were reported by Stocklgt and
Khschles [S] as mdlcatmg that there IS no re-oxidation
to feruhc acid and subsequent dlmerlzatlon at this oxlda-
tion level They reported no Increase m 3H 14C ratio,
but perhaps this can be inferred In both sets of experi-
ments, the mcorporatlon of trltmm label from the pn-
mary alcohol function of comferyl alcohol 1s compellmg
Me
OR
0
H
_I
0:
:
OMe
H__ _.
Me0
>3
: ... O
RO
OH
?Ac
Table 4 IncorporatIon of [2,-W, l-3H]-labelled comferyl alcohol mto Forsythza and
Podophyllum llgnans
Plant
Forsythia
mtermedra
Forsythia
mtermedza
Podophyllum
hexandrum
3H/4C Ratlo H/V % Incorp
Comferyl alcohol Llgnan
*g
Ratio
(C)
8 93 Arctlgenm 5 23 124 0 12
Phdlygenm 0 59 27 3 0 0048
Eplpmoresmol 1 50 364 0 029
8 93 Arctlgenm 488 16.9 0 074
Phlllygenm 014 153 00011
Epqinoresmol 109 270 0 050
8 18 Podophyllotoxm 229 12.5 0 065
Lrgnan btosynthesrs m Forsythta 1845
evidence that the coupling reaction m the biosynthesis of
these lignans does involve cmnamyl alcohol derivatives.
Synthesis of labelled compounds
[3-014CH,]Ferulic acid had been synthesized for ear-
lier Podophyllum studies [ll]. [2-4C,1-3H]Coniferyl
alcohol was obtained by mixing [2-i4C]- and [1-3H]-
labelled materials in appropriate proportions. Knoeve-
nagel condensation of [2-14C]malonic acid with acetyl-
vanillin yielded acetyl [2-14C]ferullc acid which was
converted into the acid chloride by treatment with
thionyl chloride [S]. This was reduced to the alcohol
using sodium cyanoborohydride, and deacetylation pro-
duced the required [2-14C]coniferyl alcohol in overall
yield of 30-40% from the aldehyde. [1-3H]Coniferyl
alcohol was synthestzed similarly, reducing unlabelled
acid chloride with sodium cyanoborohydride m the pres-
ence of tritrated water [17].
[i4C]Epipmoresino1 and [4C]matairesinol were pro-
duced btosynthetically by feeding L-[U-4C]phenyl-
alanine to F. intermedia tissues. The most satisfactory
source of epipmoresinol was shoots, and labelled
epipinoresinol sufficiently active for further feeding
experiments could be obtained easily by feeding labelled
phenylalanine for 72 hr as described previously (see
Table 1). An incorporation of 1 2% was obtained. These
tissues, however, produce only very low amounts of
matairesinol [4], and [4C]matairesinol was obtained
biosynthetically by use of a matairesinol4-O-glucoside-
producing cell line of F. mtermedia [lo]. A 16-day-old cell
suspension culture, taken at the beginning of the station-
ary phase when cell growth rate was declining and
matairesinol glucoside/matairesinol production was in-
creasing, was allowed to metabolize L-[U-14C]pheny-
lalanine) for three days. Matairesinol was isolated from
the extract after b-D-glucosidase treatment and thus was
obtained in good yield with high percentage incorpor-
ation (4.7%) of label.
EXPERIMENTAL
Chromatography. Prep. TLC was carned out using 0.5 mm
layers of silica gel (Merck Kiesel gel GF254); TLC zones were
eluted with Me,CO HPLC was conducted using a Sphensorb
S5 ODS2 column (250 x 4.6 mm analytical; 250 x 8 mm semi-
preparative) with a precolumn of the same packing material.
Unless stated otherwise, MeOH-H,O (9.11) was used, at flow
rates of 0.84 ml min- for analytical and 2 8 ml min- for pre-
paratrve work with UV detection at 280 nm.
Plant matenal, feeding techniques and lsolatton of hgnans. F.
mtermedia stock plants were garden grown and feeding expts
were conducted during the active growmg season of the plant,
utilizing excised shoot tips ca 4-7 cm long. Shoot tips were
immersed m Hz0 during excision, ensuring that the cut end of
the shoot was kept below the H,O surface at all times. After
transfer from the garden to the laboratory, the shoot was then
cut down to the required length and dipped into the radroactrve
precursor soln contained m small sample tubes A single shoot
was normally used for each feeding expt Phenylalanine was fed
m aq. soln, ferulic acid as the Na salt in H,O and other feedings
were conducted m DMSO-NaOH-H,O (compound drssolved
in 1 drop DMSO + 1 drop 0 5% aq. NaOH, then dil to 2 ml with
H,O) The plant was then kept at room temp. in a fume hood to
encourage uptake of the feeding soln, with dnt. H,O added as
necessary during the feeding penod At the end of the feeding
time, the plant material was cut up with scrssors, then homogen-
rzed by grinding m a mortar. The tissue was stirred wrth MeOH
(20 ml) m a 25 ml capacity Reacttvial at 70 for 1 hr. After cooling
to room temp., the crude extract was filtered by suction and the
flask washed several trmes with MeOH The comb MeOH
extracts were evapd to dryness, then Incubated with 2 ml of j-D-
glucosidase soln (2 mg ml - r m 0.1 M NaOAc buffer, pH 5)
strrring at 37 for 24 hr The mrxt. was then drl. wrth H,O (10 ml)
and extd with CH,Cl, (3 x 10 ml). The combined extracts were
evapd to dryness and the hgnan aglycones fractronated by TLC
(CH,Cl,-MeOH, 25: l), as described earlier [4].
The arctigenm-phrllygemn mrxt was purified further by TLC
[Me&O-petrol (6&80), 1.1; CHCl,-Me&O, 15.11, then
acetylated with pyndine-Ac,O (2 ml. 2 ml) at room temp. for
24 hr. The mrxt. was poured into me H,O (20 ml) and extd with
EtOAc (3 x 10 ml). The combmed extracts were washed with dil.
HCl (lo%, 3 x 10 ml), then with H,O (3 x 10 ml), dned (MgSOJ
and evapd to dryness. Lrgnan acetates were sepd by TLC [petrol
(6&80)-CH,Cl,-Me&IO, 3.1. l] and purrfied to constant sp.
act. by further TLC using Et,O, CH,Cl,-MeOH (30 l),
Me&O-hexane (1 2) and petrol (6@80qEtOAc (1: 1) If neces-
sary, arctigemn acetate was purified also by HPLC on a Spherr-
sorb S5 ODS2 semi-prep column using MeOH-H,O (1: 1).
Epipmoresmol was punfied further by TLC [Me,CO-petrol
(60-SOq, 1: 1; CHCl,-Me&O, 15.13, then converted to the
acetate as above. This was chromatographed to constant sp. act
by TLC using petrol (6&80)-CH,Cl,-Me&O, (3: 1 . 1), Et20
and CH,Cl,-MeOH (30.1).
In some cases, phillygemn acetate and eprpinoresinol diace-
tate, after punficatron by TLC using the above solvent systems,
were drl with unlabelled earner matenal to make a total wt of ca
20 mg, and were then recrystallized from MeOH ( x 4) to con-
stant sp. act. Spectral and other data for the hgnan acetates are
grven m ref. [4] The procedures used for feeding expts in
Podophyllum hexandrum plants and partial degradation of podo-
phyllotoxm are described in earlier papers [5, 6, 1 l]
Radtochemtcals DL-[ l-r4C] Phenylalanine (3 1 2 MBq mM - ),
L-[U-4C]phenylalanme (1943 GBqmM-I), [HIwater
(185 GBqml-) and [2-r4C]malomc acid as Na salt
(736 MBqmM-r) were purchased from Amersham. [3-
0r4CH,]Feruhc aad (24 4 MBq mM - ) was available from
earlier studies [ 1 l]
[2-14C]Conlferyl alcohol. Dil. aq HaSO, (0.025 M, 0.375 ml)
was added to the Na salt of [2-4C]malonlc acid (9.25 MBq,
736 MBq mM- ) and the soln left to evaporate m a vacuum
desiccator over P,Os, after which it was dissolved m Me,CO
and transferred to a Reactivral Solvent was carefully removed m
a gentle stream of N, and the residue left to dry overnight m a
desiccator over P,O,. Malomc acid (18 mg), acetyl vanillm
(40 mg), dry pyridme (0 1 ml) and freshly dust. aniline (1 ~1) were
added to the radrolabelled malomc acid and Reactivral sealed
and heated at 55 for 20 hr. The mixt. was then added to dd HCl
(lo%, 20 ml) and extracted with EtOAc (3 x 25 ml) The com-
bined extracts were evapd to dryness and the resulting acetyl
feruhc acid crystallized from aq EtOH and collected by filtration
(38 mg). A sample of unlabelled 4-O-acetyl ferulic acid produced
n-r a srmrlar manner had mp 204-205 (lit [18] 198-201)
Acetyl ferulic acrd was converted mto the and chloride by
reaction with SOCl, (0 6 ml) m a Reactrvral at 90 for 2 hr After
thus ume, the soln was evapd and the residue dned under
vacuum The chloride was dissolved m dry THF (6 ml), cooled in
an ice-bath and treated with Na cyanoborohydnde (0.32 g) A
few crystals of bromocresol green were added as an mdrcator so
that the reaction could be maintained under acidic conditrons
Sufficient HCl-THF (prepd by passing HCI gas through dry
THF) was added as necessary to maintam a yellow colour from
1846 M M A RAHMANCY al
the mdlcator [ITJ The mlxt was stlrredm the Ice-bath and after
3 hr poured mto brme (25 ml) and extracted with Et,0 (3
x 25 ml) The combmed extracts were dned (MgSO,) and then
evapd to dryness The reduction step was conducted m the dark,
as were the followmg steps mcludmg chromatography as
comferyl alcohol IS hght sensitive The resultant 4-0-acetyl
comferyl alcohol was purified by TLC (toluene-EtOAc, 1 I),
then hydrolysed m aq NaOH (1 M, 4 ml) at room temp for 1 hr
The mlxt was neutrahzed with dll HCI and the product
extracted with Et,0 (3 x 25 ml) After drymg (MgSO,), the
combined extracts were evapd to dryness yielding [2-
C]comferyl alcohol (10 mg) This was purified by TLC
(Et,O-hexane, 4 1, toluene-EtOAc, 1 1, C,H,-EtOAc, 1 1) to
constant sp act 24 7 MBq mM- , Unlabelled comferyl alcohol
synthesized m the same manner was obtamed as an 011, wrth
UVnFz nm. 291 (log& 3 65), 267 (3 96); H NMR (90 MHz,
CDCI,, TMS). 6ca 6 8(3H,brs,Ar), 65O(lH,d, J= 15 Hz, H-3),
630(1H,dt,.J=15,6Hz,H-2),430(2H,d,J=6Hz,H-l),380
(3H, s, OMe)
ceff suspension culture (6Smfj of I? rntermedia m Mg hqmd
medmm [lo] for a penod of 72 hr The culture was mamtamed
under standard con&ions (constant lllurmnatlon of 2000 lux,
rotary shaker 80 rpm, temp 25 + 1). Cells were then filtered off,
freeze-dned, powdered m a mortar and extracted with MeOH
The crude extract was hydrolysed with /?-D-glucosldase m acet-
ate buffer to liberate the aglycone as described above
Matairesmol was extracted and purified to constant sp act by
repeated TLC (CH,Cl,-MeOH, 25.1, CH,Cl,-EtOH, 5 1)
Purity was further checked by HPLC Yield 9 9 mg, sp act
3 17 MBqmM-
REFERENCES
1. Freudenberg, K and Nelsh, A C (1968) Constttutlon and
BI osynthesls of Ltgnm Springer, Berhn
2 Hlguchl, T (1985) m Btosynthesls and B~odegradatlon of
Wood Components (Higuchl, T , ed ), p I41 Academic Press,
Orlando, Flonda
[l-H]Con$eryf alcohol Acetyl feruhc acid (0 2 g) was conver-
ted mto the acid chloride using SOCl, (3 ml), usmg the reactlon
condltlons described above. At the end of the reaction, the mlxt
was evapd to dryness At the same time, tntlated Na cyano-
borohydnde was prepd by dlssolvmg Na cyanoborohydrlde
(0 5 g) and a few crystals of bromocresol green m dry THF (2 ml)
and adding sufficient HCI-THF to make the medium acldlc
Trltlated H,O (0 02 ml, 3 7 GBq) was added to this soln and the
reactlon mlxt stlrred at room temp for 3 hr, mamtammg acidic
conditions throughout 1171 Then the reaction mlxt. contaming
tntlated Na cyanoborohydnde was added to the dried acid
chloride and the reachon nuxt s&red m an Ice-bath The
product was isolated after 3 hr and purified as before Deu-
tenated 4-0-acetyl comferyl alcohol prepared m the same man-
ner had H NMR (90 MHz, CDCI,, TMS) 6 ca 6 9 (3H, br s, Ar),
650(1H,d,J=15Hz,H-3),6.20(1H,brd,5=13Hz,H-2),4.30
(lH, m, H-l), 3 80 (3H, s, OMe), 2.29 (3H, s, OAc); EIMS (probe,
70eV)m/z(rel mt) 182([M+2]+, 30%), 181 ([M-+11+,30%),
180 ([M] , 13%) The resulting trltlated acetyl comferyl alcohol
was hydrolysed and purified as before to give [l-H]comferyl
alcohol (60 mg) w&h a sp act of 95 5 MBq mM-
3. Dewlck, P M (1989) m Studres m Natural Product Chenustrj
(Rahman, A, ed ) p 459 Elsevler, Amsterdam
4 Rahman, M M A, Dewlck, P. M., Jackson, D E and Lucas,
J A (1990) Phytochemrstry 29, 1971
5 Kamll, W and Dewlck, P. M (1986) Phjtochemrstry 25,
2093
6 Jackson, D E and Dewlck, P. M (1984) Phytochemwtry 23,
1037.
7. Kamil, W and Dewick, P M. (1986) Phytochenustry 25,
2089.
K StbcIcigt, I and Khschies, M (1977j Holzjbrschung 31, 41
9 Fupmoto, H. and Hlguchl, r (19v CYbod Bes. 62, I
10 Rahman, M M A, Dewlck, P. M , Jackson, D E and Lucas,
.I A (1990) Phytochenustry 29, 1861
11 Jackson, D E and Dewlck, P M (1984) Phytochermstry 23,
1029
12 Mamtto, P., Montl, D and Gramatica, P (1974) J Chem
Sot , Per& Trans I 1727
13 Mamtto, P, Gramatrca, P and Montl, D (1975) J Chem
Sot , Perkm Trans I I 548
[*Cl Eptpmoresmol r-[LJ-C]Phenylalanme (0 925 MBq,
19.43 GBq mM- ) was fed m aq soln to each of two F mter-
me&a shoots for 72 hr as described above. Eplpmoresmol was
isolated and purified to constant sp act by repeated TLC
[Me,CO-petrol (60-80), 1 1, CHCl,-Me,CO, 15 l] Punty
was further checked by HPLC Yields of 4 03 mg, sp act.
1_04MBqmM- and 32% sp act. 13OMBqmM- were
obtained from the two shoots
14 Khschles, M, Stock@, I. and Zenk, M H (1975) I C&n
Sot , Chem Commun 879
15. Canomca, L , Gramatlca, P, Mamtto, P and Montl, D
(1979) J Chem Sot., Chem Commun 1073.
16 Gross, G. G (1985) m Bosynthesls and Blodegradatlon of
Wood Components (Hlguchl, T , ed ), p 229 Academic Press,
Orlando, Florida
17 &Xc&R F,Ben&em.,M a andDlurS&H D (1971).1 Am
Chem. Sot. 93, 2897
[VZ]Matavesmol L-[U-4C]Phenylalanme (1 85 MBq. 18 Balba, H M and Stdl, G G (1977) J Lab Cmpds Radio-
19 43 GBq mM - ) m stenle H,O (1 ml) was fed to a 16-day-old pharm 15, 309