Phenylalanme and ferulic acid are good precursors of the dibenzylbutyrolactone hgnan arctigenin and the furofuran lignans phillygenin and eptpmoresinol. Some incorporation of label from one lignan class into the other was observed. This may indicate a high degree of catabolic activny followed by non-specific mcorporation.
Phenylalanme and ferulic acid are good precursors of the dibenzylbutyrolactone hgnan arctigenin and the furofuran lignans phillygenin and eptpmoresinol. Some incorporation of label from one lignan class into the other was observed. This may indicate a high degree of catabolic activny followed by non-specific mcorporation.
Phenylalanme and ferulic acid are good precursors of the dibenzylbutyrolactone hgnan arctigenin and the furofuran lignans phillygenin and eptpmoresinol. Some incorporation of label from one lignan class into the other was observed. This may indicate a high degree of catabolic activny followed by non-specific mcorporation.
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