Stereochemical Diversity in Lignan Biosynthesis and Title Establishment of Norlignan Biosynthetic Pathway

Author(s) SUZUKI, Shiro

Wood research : bulletin of the Wood Research Institute Kyoto Citation University (2002), 89: 52-60

Issue Date 2002-09-30

URL http://hdl.handle.net/2433/53121

Right

Type Departmental Bulletin Paper

Textversion publisher

Kyoto University Review

Stereochemical Diversity in Lignan Biosynthesis . and Establishment of Norlignan Biosynthetic Pathway*l

Shiro SUZUKI*2 (Received May 31, 2002)

Keywords: lign~n, .norlignan, biosynthesis, heartwood substance, stereochemistry, Arctium lappa, Anthriscus sylvestris, Asparagus officmalls

Contents Introduction Chapter I Stereochemical diversity in lignan biosynthesis 1-1 Stereochemical diversity in lignan biosynthesis of Arctium lappa L. 1-2 Stereochemistry of lignan formation in Anthriscus OH sylvestris (L.) HofTm. OH Chapter II Establishment of norlignan biosynthetic pa­ (+)-Matairesinol (-)-Matairesinol thway II-I Pathway of norlignan biosynthesis 11-2 First enzymatic formation of the norlignan Conclusions OH Acknowledgement References OH

Introduction Lignans and norlignans are two major classes of wood extractives, accumulating specifically in heartwood OH OH composed of only dead cells and occupying the most of the trunk. These secondary metabolites are called (+)-Secoiso- (-)-Secoiso- lariciresinol lariciresinol "heartwood substances", which are synthesized in parenchyma cells and spread out from the cells to other Fig. I. Chemical structures of (+)- and (-)-enantio­ xylem elements, followed by the death of the cells. This mers of matairesinol and secoisolariciresinol. sequence of metabolic events, heartwood formation, was specific to woody plants but not to herbaceous plants. compounds. The reason woody plants are long-lived is partly because Biological activities are often related to stereochemistry they accumulate heartwood substances, some of which of compounds. Lignans are stereochemically peculiar in prevent wood-degrading fungi from rotting. natural products. In general, lignan molecules are chiral, In addition to the antimicrobial activity, lignans and and one enantiomer predominates or only one enantiomer l is present in each lignan sample isolated from plants. norlignans have various biological activities -4). Among them, the antitumor lignan, podophyllotoxin, is of special Interestingly, however, the predominant enantiomer varies interest, because it is commercially important as a staring with the plant sources. For example, optically pure, levorotatory (-)-matairesinol (Fig. 1) was isolated from material ofetoposide and teniposide, which have been used 6 as anticancer drugs in the hospital. However, the large­ Forsythia intermedia ) , while the optically pure, scaled exploitation of source plants is decreasing the dextrorotatory (+ )-matairesinol (Fig. 1) was isolated from 5 Wikstroemia sikokiana7) •(-) -Secoisolariciresinol (Fig. I) ). amount of its natural resources Therefore, it is 6 8 necessary to establish the efficient production system of from F. intermedia ) and F. koreana ) is optically pure, podophyllotoxin by which we do not need to depend on the whereas (- )-secoisolariciresinol isolated from W. sikokiana small natural resources. The studies on biosynthesis of is not optically pure [45% enantiomer excess (e.e.)]9). lignans and norlignans would afford the essential Furthermore, (+ )-secoisolariciresinol (78% e.e., Fig. 1) knowledge for biotechnological production of these was isolated from A rctium lappa petioles10). These findings strongly suggest that stereochemical control in lignan *1 This article is the abstract of Ph. D. thesis by the author formation differs among plant species. (Kyoto University, 2002). Stereochemical difference between lignan and lignin is *2 Laboratory of Biochemical Control. also of interest. Both are synthesized by one-electron

- 52 SUZUKI: Biosynthesis of Lignans and Norlignans

H3CO~~OH HO ~ OH b -----.-b I ~ ~ OCH3 OH HO HO Secoisolariciresinol Coniferyl alcohol Pinoresinol LariciresinoI

::::~:0 c d • I ...." 0 ~ OCH3 OH OH Matairesinol Arctigenin

a dirigent proteinllaccase/HzOz b pinoresinolllariciresinol reductase/NADPH c secoisolariciresinol dehydrogenase/NADP dO-methyl transferase/SAM Fig. 2. Lignan biosynthetic pathway from coniferyl alcohol to arctigenin.

I5 oxidative coupling of hydroxycinnamyl alcohols, but the presence of NADp ). fundamentally differ in optical activity; lignans are On the other hand, the recent studies in the author's optically active, whereas lignins are inactive. These laboratory have revealed that the stereochemistry oflignan results suggest that lignan biosynthesis involves biosynthesis varies with plant species. In contrast to cell­ ll stereochemically different process from that of lignin free extracts from Forsythia plants ), .Umezawa and biosynthesis. Shimada10) isolated (+ )-secoisolariciresinol (78% e.e.) Because of these important features, biosynthesis of from A. lappa petioles and the cell-free extracts catalyzed lignans and norlignans has been receiving widespread the enantioselective formation of (+ )-secoisolariciresinol interest. In this review, the author describes the recent from coniferyl alcohol in the presence of H 20 2 and findings on stereoch~micaldiversity in lignan biosynthesis NADPH. This result indicates that A. lappa has a and the establishment of norlignan biosynthetic pathway. different stereochemical control in lignan biosynthesis from that of Forsythia plants. In this chapter, the stereochemistry of lignan formation Chapter I Stereochemical diversity in lignan in two plant species other than Forsythia, e.g. Arctium lappa biosynthesis and Anthriscus sylvestris, is discussed. First, the author The first enzymatic and enantioselective formation ofan describes thatthe stereochemistry oflignan biosynthesis in optically pure lignan, (- )-secoisolariciresinol, from A. lappa is regulated organ-specificallyI6,17) In contrast achiral coniferyl alcohol with cell-free extracts from to the case of A. lappa petioles, (- )-secoisolariciresinol Forsythia intermedia in the presence of H 20 2 and NAD(P)H (65% e.e.) was isolated from seeds, and the enzyme was reported by U mezawa et al. II) They also demonstrated preparations from ripening seeds catalyzed the formation the selective oxidation of (-)-secoisolariciresinol to of (- )-secoisolariciresinol (38% e.e.) from coniferyl optically pure (- )-matairesinol III the presence of alcohol in the presence of NADPH and H 20 2. In NAD(P)6). addition, ripening seed enzyme preparation mediated the Lewis and co-workers continued to investigate the selective formation of the optically pure (>99% e.e.) (-)­ processes ofsecoisolariciresinol and matairesinol formation enantiomer of matairesinol from (± )-secoisolariciresinols in Forsythia and established the lignan biosynthetic in the presence of NADP. Second, the author describes pathway (Fig. 2). Each step, except for the final . the stereochemistry of lignan formation in A. sylvestris, a conversion from matairesinol to arctigenin12), is well herbaceous plant known to produce podophyllotoxin controlled in terms of stereochemistry; (+ )-pinoresinol is congeners and the same lignans as often found in conifer formed enantioselectively from achiral coniferyl alcohol heartwood (=heartwood lignans, e.g. yatein, hinokinin) 18). with oxidase/oxidant in the presence of dirigent protein13) . The author shows the formation of (+ )-lariciresinol (93% The formed (+ )-pinoresinol is transformed to (+)­ e.e.) and (- )-secoisolariciresinol (95% e.e.) from (±)­ lariciresinol and (- )-secoisolariciresinol with pinoresinol! pinoresinols in the presence of NADPH. This result I4 lariciresinol reductase in the presence of NADPH ), and indicates that the stereochemical property of reduction (- )-secoisolariciresinol was in turn oxidized to (-)­ catalyzed by A. sylvestris pinoresinol/lariciresinol reductase matairesinol with secoisolariciresinol dehydorogenase in (PLR) is similar to those of Forsythia PLR and A. lappa seed

53 - WOOD RESEARCH No. 89 (2002)

PLR. gave the (+ )-enantiomer of secoisolariciresinol (ca. 20% e.e.) by liquid-chromatography mass spectrometry. 1-1 Stereochemical diversity in lignan biosynthesis However, because of the incomplete separation in the of Arctium lappa L. liquid-chromatography, the value was not accurate. I7 Since Shinoda. and Kawagoye isolated arctiin, a Suzuki et al. ) determined the enantiomeric compositions glycoside of arctigenin, from seeds of Arctium lappa in 1929, of lignans, pinoresinol, lariciresinol and secoisolari­ seeds of Arctium spp. have been well-known to contain ciresinol, by gas-chromatography mass spectrometry after I significant amounts of lignans 9-23). Arctigenin isolated chiral HPLC separation; (+ )-pinoresinol (33% e.e.), from the seed was levorotatory, but the optical purity has (+ )-lariciresinol (30% e.e.) and (+ )-secoisolariciresinol I7 not been determined. Suzuki et al. ) determined the (20% e.e.) were formed with the petiole enzyme from optical purity of lignans (- )-matairesinol and (-)­ coniferyl alcohol. On the other hand, seed enzyme arctigenin by chiral high-performance liquid prepared from A. lappa ripening seeds catalyzed the chromatography (HPLC) after treating MeOH extracts formation of (- )-secoisolariciresinol (38% e.e.), (- )­ from the seeds with f3-g1ucosidase. Both lignans'were pinoresinol (22% e.e.), and (-)-lariciresinol (>99% e.e.) optically pure (>99% e.e.). This is in good accordance from coniferyl alcohol in the presence of NADPH and I6 with previous reports; all the dibenzylbutyrolactone H 20 2 ). The enzymatic experiments with coniferyl lignans of which enantiomeric compositions have so far alcohol exhibited the stereochemical diversity, which is in been determined precisely by chiral HPLC are optically line with the discordance of the predominant enantiomers 24 pure ) of secoisolariciresinol isolated from different organs of A. In addition, small amounts of secoisolariciresinol were lappa (Fig. 3). isolated from seeds. In contrast to (+ )-seoisolariciresinol Pinoresinol/lariciresinol reductase (PLR), responsible IO (78% e.e.) isolated from the petioles ), (-)­ for reduction of pinoresinol to lariciresinol, and secoisolariciresinol (65% e.e.) was isolated from MeOH lariciresinol to secoisolariciresinol, was purified from I6 I4 extract of the seeds after glucosidase treatment ). Acid Forsythia intermedia ) , and this enzyme was detected from 5 26 hydrolysis (H2S04) also yielded (- ~-secoisolariciresinol Zanthoxylum ailanthoidei ) and Daphne odora ). Incubation (82% e.e.) from the MeOH extractsI ). This is the first of (±)-pinoresinols with the seed enzyme preparation example that different enantiomers of a particular lignan yielded almost optically pure (-)-secoisolariciresinol occur predominantly in different organs of a single plant (99% e.e.) and (+ )-lariciresinol (85% e.e.), and (-)­ species, indicating the·· stereochemical diversity of lignan secoisolariciresinol (91 % e.e.) was formed from (±)­ biosynthetic mechanism in A. lappa. lariciresinols with the enzyme preparation 17). Thus, the U mezawa and Shimada10) reported that the incubation seed enzyme preparation had PLR activity. The of coniferyl alcohol with the petiole enzyme preparation predominant formation of (- )-secoisolariciresinol from

H 0H OH 2r HO OH H1I1I8'° ,,:::::,: I + ~I"'::""" 0 . OH Petiole HOY OCH3 (+)-Secoiso­ (+)-Pinoresinol (+)-Lariciresinol lariciresinol /" [20% e.e.] [33% e.e.] [30%e.e.]

OCH3 OH

Coniferyl enzyme alcohol

HO HO

(-)-Secoiso­ (-)-Pinoresinol (-)-Lariciresinol laricires~ol [22% e.e.] [>99% c.c.] [38% e.e.] Fig. 3. Formation ofpinoresino1, lariciresinol and secoisolariciresinol by Arctium lappa enzyme preparations. The petiole enzyme preparation catalyzes the lignan formation in favor of (- )-enantiomer, while the seed enzyme preparation does in favor of (+ )-enantiomer.

- 54- SUZUKI: Biosynthesis of Lignans and Norlignans these racemic lignans are in line with the results of ciresinol. Formation of optically pure lignan is finally I6 incubation of coniferyl alcohol with the seed enzyme ). achieved in the conversion of secoisolariciresinol to The petiole enzyme also exhibited PLR activity giving matairesinol in this plant species. rise to lariciresinol and secoisolariciresinol from (±)­ Taken together, there is a great stereochemical diversity pinoresinols, and secoisolariciresinol from ( ± )­ in lignan biosynthesis, and not only the enantioselective lariciresinols17). Interestingly, however, the predominant coupling of coniferyl alcohol assisted by dirigent protein enantiomers of the product lignans, (- )-secoisola­ but also the subsequent several steps must play substantial riciresinol and (+ )-lariciresinol, formed from (±)­ roles in production of optically pure lignans. pinoresinols and (±)-lariciresinols were the same as those obtained with the seed enzyme, and (- )­ 1-2 Stereochemistry of lignan biosynthesis in secoisolariciresinol is opposite to the predominant Anthriscus sylvestris (L.) Hoffm. enantiomer, (+ )-secoisolariciresinol, isolated from the Due to the limited supply of podophyllotoxin (Fig. 4) 5 petiole. The enantiomer excess values ofthe formed (-)­ from natural resources ), the alternative sources have been secoisolariciresinol (44 and 37% e.e.) were much lower searched. Anthriscus sylvestris (L.) Hoffm. might be one of than those formed with the seed enzyme which are almost the candidates, because it produces angeloyl 30 optically pure (99 and 91 % e.e.). podophyllotoxin ) as well as significant amounts of a 31 36 These results can be accounted for by postulating that A. precursor of podophyllotoxin, deoxypodophyllotoxin - ) lappa has PLR isoforms showing different selectivity in (Fig. 4). In addition, it produces the typical heartwood 34 terms of the substrates, (+ )-pinoresinol and (-)­ lignans yatein and hinokinin ) (Fig. 4), which are found pinoresinol. Although final conclusions await further specifically in the heartwood region in the conifers 7 38 experiments, this view is in good accordance with recent Libocedrus yateensii ) and Chamaecyparis obtusa ) findings on PLR of different plant species as follows: PLR respectively. Thus, A. sylvestris is probably a good plant was partially purified from Forsythia intermedia cv. material for lignan biosynthesis studies to access Lynwood gold27 ), and its cDNAs were cloned and mechanisms involved in antitumor and heartwood lignan I4 expressed in E. coli ). Both plant and recombinant formation. As the first step, it is necessary to characterize proteins exhibited the same stereochemical property; each the precursor lignans of yatein and deoxypodophyllotoxin protein catalyzed selective formation of (+ )-lariciresinol in A. sylvestris. 18 and (- )-secoisolariciresinol from (± )-pinoresinols, and Suzuki et al. ) preliminarily surveyed lignans in the {3­ (- )-secoisolariciresinol from (± )-lariciresinols in the glucosidase-treated MeOH extracts of both aerial parts I4 5 presence ofNADPH ). PLR of Zanthoxylum ailanthoidei ) and roots of A. sylvestris. GC-MS analysis revealed that also showed similar stereochemical selectivity to Forsythia the presence of the lignans, yatein and secoisolariciresinol. PLR. In contrast, PLR activity from Daphne genkwa In addition, nemerosin and deoxypodophyllotoxin, which which exhibited the opposite stereochemical property to were previously isolated from Anthriscus Spp.3I--:36,39), were 39 the Forsythia and Zanthoxylem PLRs; the Daphne crude identified by comparing their spectrometoric data ,40). enzyme preparation catalyzed selective formation of (-)­ After fractionating the f3-glucosidase-treated MeOH 26 lariciresinol (23% e.e.) from (± )-pinoresinols ). These extracts of A. sylvestris by silica gel column results indicated that different PLRs which have opposite chromatography, lariciresinol, matairesinol, hinokinin, stereochemical properties with respect to lariciresinol and pluviatolide, and bursehernin (Fig. 4) were identified by secoisolariciresinol formation distribute in different plant GC-MS analysis18). Secoisolariciresinol, lariciresinol, species. Furthermore, the presence of cDNAs cor­ matairesinol, pulviatolide and brusehernin were identified responding to the two stereochemically distinct PLRs in a for the first time in Anthriscus spp. 28 single plant species was demonstrated by Fujita et al. ) When coniferyl alcohol was incubated with the A. although they did not mention the physiological roles ofthe sylvestris enzyme preparation in the presence of H 20 2 and two isoforms. On the other hand, the author's present NADPH, the lignans, pinoresinol, lariciresinol, and results strongly suggest that the two PLR isoforms are secoisolariciresinol, were formed. Furthermore, the expressed differentially in A. lappa. enzymatic conversion of (± )-pinoresinol to (+)­ As for the stereochemistry of pinoresinol formation, lariciresinol (93% e.e.) and (- )-secoisolariciresinol (95% dirigent protein has not yet been isolated from A. lappa. e.e.) by PLR was demonstrated in the presence of I8 However, a recent detection of a dirigent-protein-like gene NADPH ). 9 from A. lappa using a PCR-guided strategl ) suggests that The PLR activity together with enzymatic formation of stereochemistry of formation of pinoresinol from coniferyl the lignans from coniferyl alcohol accorded well with those alochol in A. lappa is also under control ofdirigent protein. with A. lappa and Forsythia spp6,8,11,27,41,42). In addition, In accordance with the presence oflarge amounts of two the PLR-catalyzed selective formation of (+ )-lariciresinol optically pure dibenzylbutyrolactone lignans, (- )­ and (- )-secoisolariciresinol from (± )-pinoresinols with matairesinol and (-)-arctigenin, in A. lappa seeds, the A. sylvestris enzyme preparation suggested that the secoisolariciresinol dehydrogenase activity was detected in stereochemical property of A. sylvestris PLR-catalyzed the seed enzyme preparation which gave rise to optically reduction was similar to those of Forsythia PLR27 ) and A. pure (- )-matairesinol following incubation of racemic lappa ripening seed PLR17) I7 (± )-secoisolariciresinols in the presence of NADp ). The lignan formation by the Anthriscus enzyme Thus, although formation of secoisolariciresinol is preparation along with the detection of lariciresinol and controlled stereochemically, the control is not enough secoisolariciresinol from the plant suggests strongly that strong to produce only one enantiomer of secoisolari- the conversion, pinoresinol-lariciresinol-secoisolaricire-

55 - WOOD RESEARCH No. 89 (2002)

OH

Lariciresinol Secoiso­ Matairesinol lariciresinol OOh OH

o Rl=OCH3, R2=CH3, Yatein Hinokinin Nemerosin Rl=H, R2=H, Pluviatolide ­ Rl=H, R2=CH3, Bursehemin

Deoxypodo­ phyllotoxiri Podophyllotoxin Fig. 4. Chemical structures of podophyllotoxin and Anthriscus sylvestris lignans. sinol. H~OH Chapter II Establishment of norlignan biosynthetic patit.way Typical norlignans having the I, 3-diphenylpentane (E)-Hinokiresinol [C6-C3(C2)-C6] structure le.g. hinokiresinol [(E)-hino­ kiresinol], agatharesinol, and sequirin-C, Fig. 51 occur in HO OH coniferous trees (especially in heartwood) ofCupressaceae, Taxodiaceae, and Araucariaceae43-46), while y-lactonized OH 1, 3..;diphenylpentane norlignans (e.g. pueroside A and B) were isolated from two Leguminosae trees (Pueraria lobata and Sophora japonica)47-49). Some monocotyledonous OH OH Liliaceae and H ypoxidaceae plants are also good sources of Agatharesinol Sequirin-C 1,3-diphenylpentane and 1,5-diphenylpentane norli­ gnans. For instance, (Z)-hinokiresinol (=nyasol) (Fig. 5) RI0~O-o-OR2 which is the geometrical isomer of a coniferous heartwood norlignan, (E)-hinokiresinol, was isolated from Asparagus Asparenydiol: R1=H, R2=H 3 and Anemarrhena ,50,51). Asparenyol: Rl=H, Ri=CH3 It is well known that norlignans accumulate specifically Asparenyn:R1=CH3,R2=CH3 in conifer heartwood. Heartwood coloration of C.japonica Fig. 5. Chemical structures of norlignans and related (Japanese cedar)52,53) and Chamaecyparis obtusa (hinoki compounds. cypress)54) is due to norlignans. The- normal heartwood coloration of C. japonica and C. obtusa is pale salmon pink, which has been appreciated in Japan. However, black­ attention has been paid to regulating the norlignan discoloration often occurs in C. japonica heartwood, which biosynthesis. lowers the valueofthe wood. To solve the problem, much Several hypothetical pathways for norlignan biosyn-

56 SUZUKI: Biosynthesis of Lignans and Norlignans thesis had been proposed based on the chemical structures OH of norlignans37,43,55--60). First, Enzell and Thomas58) HO suggested the coupling of two phenylpropane units 6&,O'I':OH followed by a loss of one carbon atom giving rise to [l3C]Cinnamic agatharesinol. Later, a coupling of 4-coumaric acid with 5 ~ 3 4-coumaryl alcohol that involved the loss of the carbon 4 acids (Z)-Hinokiresinol atom at the 9-position of 4-coumaric acid was proposed Fig. 6. 13C-Labelling patterns of (Z)-hinokiresinol 13 13 independently by Birch and Liepa59), and Beracierta and incorporating [7- C]cinnamic acid, [8_ C] 13 Whiting60) Erdtman and Harmatha37 ) subsequently cinnamic acid, or [9- C]cinnamic acid. 7­ 13 13 ....= assumed that C8-C8' linked lignans formed by the coupling 13C; .=8_ C; .=9_ C. of two phenylpropanoid monomers were converted to norlignans via an intramolecular rearrangement of the side individually, and the 13C-enriched position III the side chain of the carbon skeleton. Despite these proposals of chain of (Z)-hinokiresinol was determined by GC-MS and 13 coupling modes of two phenylpropane units, none of them 13C NMR. When [7- C]cinnamic acid was adminis­ were supported by any concrete experimental evidence. tered, specific 13C enrichments at C-l (11.7 atom% excess) In this chapter, the establishment of norlignan and C-3 (10.6 atom% excess) of (Z)-hinokiresinol were biosynthetic pathway is described. Using a fungal­ observed. Similarly,13C enrichments at C-2 (32.3 atom% elicited Asparagus officina/is cell system, it has been excess) and C-4 (31.2 atom% excess) occurred when [8­ 13 demonstrated that (Z)-hinokiresinol originates from two 13C ]cinnamic acid was fed. As for the feeding of [9_ C] non-identical phenylpropanoid monomer: 4-coumaryl cinnamic acid, significant 13C enrichment at only C-5 (26.3 alcohol and a 4-coumaroyl compound61 ). Furthermore, atom% excess) was observed. 13C enrichments at other the first in vitro norlignan formation with an enzyme positions were negligible (-0.27-0.56 atom% excess). preparation has been demonstrated62 ). The enzyme These 13C-tracer experiments unequivocally established preparation from fungal-elicited A. officina/is cells catalyzed that all 17 carbon atoms of (Z)-hinokiresinol are derived the formation of (Z)-hinokiresinol from two non-identical from phenylpropanoid monomers. Also, it was phenylpropanoid monomers, 4-coumaryl alcohol and 4­ conclusively demonstrated that the side chain, 7-C, 8-C, coumaroyl CoA, and from a dimer, 4-coumaryl 4­ and 9-C atoms ofcinnamic acid were incorporated into C-l coumarate, without any additional cofactors. Based on and C-3, C-2 and C-4, and C-5 of (Z)-hinokiresinol, the results of the enzymatic reaction, the novel biosynthetic respectively (Fig. 6). Thus, intramolecular rearrange­ mechanism for (Z)-hinokiresinol via the ester enolate ment of the side chain carbon atoms of the monomers did Claisen rearrangement is proposed. not occur in (Z)-hinokiresinol formation. 61 Suzuki et a/. ) next demonstrated the immediate II-I Pathway of norlignan biosynthesis precursor(s) in (Z)-hinokiresinol formation. First, they 63 Within the last decade, Takasugi ) reported that a synthesized the following 13C and/or 2H labelled 13 herbaceous plant, Asparagus officina/is, inoculated with a compounds, 4-[ring- C 6 ]coumaric acid, 4-[9, 9-2H2, ring­ 2 phytopathogen produced (Z)-hinokiresinol (Fig. 5) as a 13C6]coumaryl alcohol, 4-[7,9, 9- H 3]coumaryl alcohol, 64 13 phytoalexin. Terada et a/. ) reported that cell cultures and 4-[9-2H , ring- C6]coumaraldehyde, and then of the plant produced norlignan-related C6-C5-O-C6 administered the compounds individually to the elicited compounds, asparenydiol and its methylated compounds Asparagus cells. 13 (asparenyol and asparenyn) (Fig. 5), without any elicitor When 4-[ring- C 6 ]coumaric acid was fed, GC-MS treatment. Later, they demonstrated that asparenyol was analysis of the formed (Z)-hinokiresinol showed the derived from two units of phenylalanine with a loss of one significant enhancement of ion peak at m/z 408 carbon atom at the 9-position of phenylalanine based ([M] + + 12), indicating that 4-coumaric acid was on the l3 65 on C tracer experiments ), and assumed hinokiresinol as metabolic pathway leading to (Z)-hinokiresinol. When 4­ 2 13 a putative precursor of asparenyol, although without any [9,9- H 2, ring- C 6 ]coumaryl alcohol was fed to the cells, 66 experimental evidence ). great enhancement of ion peak at m/z 410 was observed. 61 Suzuki et a/. ) established a A. officinalis cell system This result demonstrated that two units of 4-coumaryl producing a norlignan, (Z)-hinokiresinol (yield: 0.02% alcohol were converted ultimately to (Z)-hinokiresinol with based on dried cell weight). They isolated (Z)­ the loss of the two 9-positioned deuterium atoms from one hinokiresinol from MeOH extract of elicitor-treated A. of the monomers, but did not imply that two units of the officina/is cells and identified it by NMR analysis. alcohol were directly involved in dimerization giving rise to Next, L- [ring- 13C6] phenylalanine was administered to (Z) -hinokiresinol. 2 I the elicitor-treated A. officina/is cells, and the fi-glucosidase­ Importantly, when 4-[9,9- H 2, ring- 3C6]coumaryl treated MeOH extract was submitted to GC-MS analysis alcohol was administered, enhancement at m/z 404 to examine the incorporation of 13C . Compared with the ([M] + + 8) was also observed, which was assigned to (Z)­ mass spectrum of unlabelled (Z)-hinokiresinol TMS ether [2H2, 13C6]hinokiresinol TMS ether, i.e. the product of 2 13 ([M]+=m/z 396), the enhanced ion peak at m/z 408 coupling of one unit of exogenous 4-[9,9- H 2, ring- C 6 ] ([M] + + 12) was observed, indicating unequivocally that coumaryl alcohol with an endogenous unlabelled two aromatic rings of (Z)-hinokiresinol were derived from phenylpropane unit. This endogenous precursor-induced L-phenylalanine. dilution effect is rather common in feeding experiments, Similarly, cinnamic acids labelled with 13C at the side and, in fact, also occurred in the case of L-[ring_ 13C6] chain were next administered to the Asparagus cells phenylalanine administration. In addition to the

57 WOOD RESEARCH No. 89 (2002) significant enhancement of the ion peak at m/z 408 R 9 0 OH 3 ([M]+ + 12); due to the incorporation of two eC6] phenylalanine units into (Z)-hinokiresinol, great enhancement was also observed at m/z 402 ([M] ++6), and - '?' . . 3 - ~I may be ascribed to coupling of one eC6]phenylalanine ~ unit and one endogenous unlabelled phenylpropane unit. L-Phenyl­ Cinnamic Interestingly, however, the ion peak at m/z 402 ([M]++6, OH 2 alanine acid 4-Coumaryl (Z)-[13C6]hinokiresinol TMS ether) after 4-[9, 9- H 2, ring­ alcohol 13C6]coumaryl alcohol' administration was not significant. If one such labelled 4-coumaryl alcohol unit and one endogenous unlabelled 4-coumaryl alcohol unit are directly involved in the dimerization, both [M] + + 8 and [M] + + 6 ions must appear with equal intensity. This suggests that two 4-coumaryl alcohol units were not involved directly in coupling, and that the coupling of one 4-coumaryl alcohol unit and another phenylpropane' unit which can be formed from 4-coumaryl alcohol. It is established that the reduction of cinnamaldehyde (Z)-Hinokiresinol and cinnan;l:Oyl CoA by cinnamyl alcohol dehydrogenase Fig. 7. Proposed biosynthetic pathway for (Z)­ (CAD) and cinnamoyl CoA reductase (CCR), respectively, hinokiresinol. The C6-C3 moiety of (Z)­ 67 is reversible -69). Hence, it was hypothesized that some hinokiresinol was derived from 4-coumaryl 13 alcohol, while the C6-C2 moiety was from a 4­ of the exogenously administered 4-[9, 9-2H2, ring_ C6] 13 coumaroyl compound (4-coumaric acid, R OH; coumaryl alcohol were converted to 4-[9-2H , ring_ C6 ] 13 4-coumaroyl CoA, R=SCoA ; 4-coumaral­ coumaraldehyde and 4-[ring_ C6] coumaroyl CoA, which 13 dehyde, R=H). in turn coupled with 4-[9, 9-2H2, ring_ C 6 ]coumaryl alcohol to afford (Z)-[2H2, 13C12]hinokiresinol. from phenylpropanoid monomers with the loss of one To test this hypothesis, the simultaneous administration carbon atom at the 9-position of one of the monomers. of two distinct, possible precursors was carried out61 ). 13 The C6-C3 moiety of (Z)-hinokiresinol is originated from Thus, equal molar amounts of 4-[ring_ C ]coumaric acid 6 4-coumaryl alcohol, while. the C -C2 moiety is from a and 4-[7,9, 9-2H ]coumaryl alcohol were administered to 6 3 4-coumaroyl compound. elicited cells in a single flask, and the results were compared with those obtained after individual 11-2 First enzymatic formation of the norlignan 62 administration of the two precursors as positive controls. Suzuki et al. ) demonstrated in vitro norlignan Administration of4-[7,9,9-2H3]coumaryl alcohol alone formation for the first time. Thus, an enzyme preparation resulted in formation of (Z)-eH4]hinokiresinol TMS ether from fungal-elicited Asparagus officinalis cultured cells ([M]+ +4) and (Z)-eH3]hinokiresinol TMS ether catalyzed the formation of a norlignan, (Z)-hinokiresinol, ([M]+ +3) which corresponded to (Z)-eH2, 13C12] from two non-identical phenylpropanoid monomers, 4­ hinokiresinol TMS ether ([M]+ + 14) and (Z)-eH2, 13C6] coumaryl alcohol and 4-coumaroyl CoA, and from a dimer, hinokiresinol TMS ether ([M]+ +8), respectively, in the 4­ 4-coumaryI4-coumarate, without any additional cofactors. 13 [9, 9-2H2, ring_ C 6 ]coumaryl alcohol administration. Proof that the formation of (Z)-hinokiresinol was 13 Similarly, administration of only 4-[ring- C 6 ]coumaric enzymatic was obtained by control experiments; the acid resulted in the enhanced ion peaks of [M] + + 12 I(Z)­ formation of (Z)-[2H3]hinokiresinol from 4-[7, 9, 9-2H3] 3 eC 12]hinokiresinol TMS etherl as already described. coumaryl alcohol and 4-coumaroyl CoA did not occur In sharp contrast, the simultaneous administration of the when the denatured enzyme preparation was used, and two precursors provided no significant evidence in barely occurred when the enzyme preparation or the 2 coupling products of two units of 4-[7,9, 9- H 3]coumaryl substrate(s) were omitted from the complete assay. On alcohol ([M]+ +4, (Z)-eH4]hinokiresinol TMS ether). the other hand, incubation of 4- [7, 9, 9-2H3]coumaryl In addition, the ion peak at m/z 408 ([M]+ + 12, (Z)­ alcohol and unlabelled 4-coumarate with the enzyme 13 [ C 12] hinokiresinol TMS ether) showed only a small preparation did not afford (Z)-[2H3]hinokiresinol, increase, compared with the unlabelled one. The ion eliminating the mechanism that 4-coumaroyl CoA was first peak at m/z 405 ([M]++9) was prominent, and was hydrolyzed to 4-coumarate, which coupled with 4­ 2 derived by coupling one 4-[7,9, 9- H 3]coumaryl alcohol coumaryl alcohol to afford (Z)-hinokiresinol. These 13 unit with one 4-[ring_ C6 ]coumaric acid unit, confirming results demonstrate for the first time a norlignan synthase our hypothesis that (Z)-hinokiresinol is not formed by the activity. direct dimerization of two units of 4-coumaryl alcohol. Since esters are often biosynthesised by acyltransferase­ Instead, the C6-C3' moiety of (Z)-hinokjresinol, is derived catalyzed condensation between the corresponding CoA 70 62 from 4-coumaryl alcohol unit, while the C6-C2 moiety is esters and alcohoI ), Suzuki et al. ) next hypothesized that from a 4-coumaroyl compound (HO-C6H5-CH=CH­ (Z)-hinokiresinol was formed via the ,coupling of 4­ CO-R) such as 4-coumaric acid, 4-coumaroyl CoA, or coumaryl alcohol and 4-coumaroyl CoA to afford 4­ 4-coumaraldehyde .(Fig. 7). coumaroyl 4-coumarate followed by C7-C8'·, bond In conclusion, it has been shown for the first time that all formation and C9' decarboxylation (Fig. 8). To test this 2 carbon atoms ofa norlignan, (Z)-hinokiresinol, are derived hypothesis, 4-[7,9, 9- H 3]coumaryl 4-coumarate was

58 SUZUKI: Biosynthesis of Lignans and Norlignans

o enzyme. These results suggest that the great diversity in 4-Coumaroyl SCoA enantiomeric compositions of lignans among plant species CoA are at least partly due to the differential expression of PLR isozymes which have distinct stereochemical selectivity. + The biosynthetic pathway for a norlignan, (Z)­ HO Esterification OH hinokiresinol, was proposed. Thus, the coupling of 4-coumaryl alcohol with 4-coumaroyl CoA afforded (Z)-hinokiresinol. In addition, the enzyme activity ~ /; 4-Coumaryl forming (Z)-hinokiresinol from 4-coumaryl alcohol and alcohol 4-coumaroyl CoA was detected. In this reaction process, HO 4-coumaryl 4-coumarate is probably an intermediate ~8'bond compound, and in fact, the compound was transformed to ~ / ~~~tion/ (Z)-hinokiresinol enzymatically. These results strongly : I if_ " decarboxylation suggest the following mechanism for (Z)-hinokiresinol biosynthesis: (i) 4-coumaryl alcohol couples with 4­ ~ coumaroyl CoA to afford 4-coumaryl 4-coumarate. OH OH (ii) 4-Coumaryl 4-coumarate are transformed to (Z)­ (Z)-Hinokiresinol hinokiresinol via the ester enolate Claisen rearrangement and subsequent decarboxylation. Fig. 8. A putative mechanism for the formation of (2)­ hinokiresinol with A. officinalis enzyme prepara­ tion. Acknowledgement The author wishes to thank Associate Professor Dr. administered to the fungal-elicited A. oJJicinalis cells. 4­ Toshiaki Umezawa, Wood Research Institute, Kyoto Coumaryl 4-coumarate is found to be efficiently converted University, for many helpful discussions and critical to (Z)-hinokiresinol. reading of the manuscript. Furthermore, the conversion of 4-coumaryl 4-coumarate to (Z)-hinokiresinol was also demonstrated by an in vitro 62 References experiment ); incubation of 4-[7, 9, 9- 2H3Jcoumaryi 4­ coumarate with the enzyme preparation resulted in 1) Y. IIDA, K.-B. OH, M. SAITO, H. MATSUOKA and H. enzymatic (Z)-[2H3Jhinokiresinol formation in a high KURATA: Planta Med., 66, 435-438 (2000). yield. These in vivo and in vitro experiments strongly 2) T. YAMADA: Bull. For. & For. Prod. Res. Inst., 69-162 (1998). suggest that 4-coumaryl 4-coumarate is the intermediate 3) H.A. OKETCH-RABAH, S.F. DOSSAJI, S.B. CHRISTENSEN, K. FRYDENVANG, E. LEMMICH, C. CORNETT, C.E. OLSEN, M. between the phenylpropanoid monomers (4-coumaryl CHEN, A. KHARAZMI and T. THEANDER: J. Nat. Prod., 60, alcohol and 4-coumaroyl CoA) and (Z)-hinokiresinol (Fig. 10 17-1022 (1997). 8). 4) E. MINAMI, M. TAKI, S. TAKAISHI, Y. hJIMA, S. TSUTSUMI Incubation of 4-coumaryl 4-coumarate with horseradish and T. AKIYAMA: Chem. Pharm. Bull., 48, 389-392 (2000). peroxidase in the presence of H 20 2 did not afford (Z)­ 5) C. CANEL, R.M. MORAES, F.E. DAYAN and D. FERREIRA: hinokiresinol, indicating the peroxidase does not initiate Phytochemistry, 54, 115-120 (2000). the C7-C8' bond formation and ultimate decarboxylation. 6) T. UMEZAWA, L.B. DAVIN and N.G. LEWIS: J. BioI. Chem., This is in sharp contrast to C8-C8' bond formation 266, 10210-10217 (1991). between hydroxycinnamyl alcohol units in lignan and 7) T. UMEZAWA and M. SHIMADA: Mokuzai Gakkaishi, 42, lignin biosynthesis which is mediated by peroxidase or 180-185 (1996). 71 laccase ). Instead, 4-coumaryl 4-coumarate might be 8) T. UMEZAWA, H. KURODA, T. ISOHATA, T. HIGUCHI and M. converted to (Z)-hinokiresinol via the ester enolate Claisen SHIMADA: Biosci. Biotechnol. Biochem., 58, 230-234 (1994). rearrangement (the [3,3J-sigmatropic rearrangement of 9) T. OKUNISHI, T. UMEZAWA and M. SHIMADA: J. Wood Sci., 46, 234-242 (2000). allyl esters to y, 8-unsaturated carboxylic acids) 72) 10) T. UMEZAWA and M. SHIMADA: Biosci. Biotechnol. Biochem., followed by decarboxylation. However, the true 60, 736-737 (1996). mechanism awaits further experiments with purified 11) T. UMEZAWA, L.B. DAVIN and N.G. LEWIS: Biochem. enzymes. Biophys. Res. Commun., 171, 1008-1014 (1990). 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59 WOOD RESEARCH No. 89 (2002)

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