Phytochemistry Reviews 2: 371390, 2003. 2004 Kluwer Academic Publishers. Printed in the Netherlands.

371

Diversity in lignan biosynthesis

Toshiaki Umezawa
Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan (E-mail, tumezawa@kuwri.kyoto-u.ac.jp)

Key words: biosynthesis, evolution, lignans, phylogenetic distribution, stereochemistry, structural diversity

Abstract Lignans are phenylpropanoid dimers, where the phenylpropane units are linked by the central carbon (C8) of their side chains. Ligans vary substantially in oxidation level, substitution pattern, and the chemical structure of their basic carbon framework. In addition to structural diversity, lignans show considerable diversity in terms of enantiomeric composition, biosynthesis, and phylogenetic distribution. In this review, these diversities are outlined and the phylogenetic distribution of plants producing 66 typical lignans is listed. The distribution is correlated with the putative biosynthetic pathways of the lignans and discussed from evolutionary aspects. Abbreviations: SIRD Secoisolariciresinol dehydrogenase; PLR pinoresinol lariciresinol reductase; DP dirigent protein Introduction Lignans, constituting an abundant class of phenylpropanoids, have been receiving widespread interest in many elds. This is mainly because these compounds have a number of medically important biological activities, e.g. antitumor, antimitotic, and antiviral properties (MacRae and Towers, 1984; Ayres and Loike, 1990; Umezawa, 1996), as well as unique stereochemical properties (Umezawa, 1997; Lewis and Davin, 1999; Umezawa, 2001). During the last decade, signicant advances have been made in the chemistry and biosynthesis of lignans (Umezawa, 1997; Lewis and Davin, 1999; Umezawa, 2001). Additionally, the structural, enantiomeric, and biosynthetic diversity exhibited by lignans, as well as their diverse phylogenetic distribution have been noticed (Umezawa, 2001). Lignan biosynthesis has been found to be closely related to but distinct from those of other phenylpropanoids, such as norlignans, lignins, and neolignans (Umezawa, 2001; Suzuki et al., 2001; Suzuki et al., 2002a). The diversity exhibited by lignans and the similarity of their biosynthesis to those of other phenylpropanoids piqued our scientic interest in the origination and evolution of phenylpropanoid biosynthesis in vascular plants, a central theme in plant science. Lignans are distributed widely in vascular plants. But, unlike lignins, they are not ubiquitous. Hence, lignans may be a good subject for studying the evolution of plant secondary metabolism. Herein the author outlines the various diversities of lignans and correlates them with a phylogenetic classication of lignan producing plants. Nomenclature The term, lignan, was introduced by Haworth (1936) to describe a group of phenylpropanoid dimers, where the phenylpropane units were linked by the central carbon (C8) of their propyl side chains (Figure 1). McCredie et al. (1969) proposed to extend the term to cover all natural products of low molecular weight that arise primarily from the oxidative coupling of phydroxyphenylpropene units, while Gottlieb (1972) coined the term neolignan for compounds composed of two phenylpropane units linked in a manner other than C8-C8 . However, later neolignans were redened as the dimers of allyl- and/or propenylphenyl monomers, while lignans were regarded as the dimers of cinnamyl alcohols and/or cinnamic acids (Gottlieb, 1978). In spite of these redenitions, Haworths denition of lignans (Haworth, 1936) and Gottliebs former

372 denition of neolignans (Gottlieb, 1972) have been used widely, and recently were adopted by the IUPAC recommendations 2000 (Moss, 2000). Analogs of lignans composed of three and four phenylpropane units have been commonly called sesquilignans and dilignans, respectively (Umezawa, 2000). However, the most recent IUPAC recommendations named these compounds sesquineolignans and dineolignans, respectively. The new terms are logical, because these tri- and tetramers involve inherently neolignan type linkages. This review follows the IUPAC recommendations (Moss, 2000). (-)-secoisolariciresinol (-)-23 (dibenzylbutane); (-)matairesinol (-)-27, (-)-arctigenin (-)-28, (-)-hinokinin (-)-29, (-)-pluviatolide (-)-30, (-)-kusunokinin (-)67, (-)-haplomyrfolin (-)-68, thujaplicatin methyl ether (-)-70, and (-)-4-demethylyatein (-)-71 (Charlton and Chee, 1997) (dibenzylbutyrolactone); and ()-wikstromol (= nortrachelogenin) (-)-33 and (-)methyltrachelogenin (-)-69 (Khamlach et al., 1989) (8-hydroxydibenzylbutyrolactone). Naturally occurring lignans have been found to exist exclusively as one enantiomer or as enantiomeric mixtures with various enantiomeric compositions (% e.e. values) (Table 1). Sometimes even racemic lignans have been found to occur (Umezawa et al., 1997a). There is no example of furofuran and furan lignans proven to be optically pure by chiral HPLC analysis (Umezawa et al., 1997a). For instance, pinoresinol 1 isolated from Wikstroemia sikokiana was found to be a mixture of both enantiomers with 74% e.e. [()>(+)] (Umezawa and Shimada, 1996a), and that from Larix leptolepis was dextrorotatory (92% e.e.) (Nabeta et al., 1991). In contrast, all dibenzylbutyrolactone lignans which have so far been analyzed by chiral HPLC have been found to be optically pure (>99% e.e.) regardless of the sign of optical rotation (Table 1) (Umezawa et al., 1997a). As for dibenzylbutane lignans, secoisolariciresinol 23 from Forsythia plants is optically pure and levorotatory (Umezawa et al., 1991; Umezawa et al., 1992) and that from Phyllanthus sp. is almost optically pure (98% e.e.) in favor of the (+)enantiomer (Umezawa et al., 1997b). On the other hand, this lignan isolated from W. sikokiana and Arctium lappa (petiole) is not optically pure, with 45% e.e. [(-)>(+)] (Okunishi et al., 2000) and 81% e.e., (+)>(-) (Umezawa and Shimada, 1996b; Suzuki et al., 1998; Suzuki et al., 2002a), respectively (Table 1). Umezawa et al. (1997a) also pointed out that the predominant enantiomers (or the signs of optical rotation) of furofuran, furan, and dibenzylbutane lignans vary with plant species (Table 1). For example, (-)-secoisolariciresinol (-)-23 was isolated from many plants, e.g. W. sikokiana (45% e.e.) (Okunishi et al., 2000), Forsythia spp. (optically pure) (Umezawa et al., 1991; Umezawa et al., 1992), Xanthoxylum ailanthoides (Ishii et al., 1983), and Araucaria angustifolia (Fonseca et al., 1978),while (+)-secoisolariciresinol (+)-23 was obtained from Phyllanthus sp. (98% e.e.) (Umezawa et al., 1997b) and Daphne spp. (>9997% e.e.) (Okunishi et al., 2001) (Table 1). Similarly, as shown in Table 1 the predominant enantiomers of pinoresinol and lar-

Structural diversity of lignans Lignans are classied into the following eight subgroups based upon the way in which oxygen is incorporated into the skeleton and the cyclization pattern: furofuran, furan, dibenzylbutane, dibenzylbutyrolactone, aryltetralin, arylnaphthalene, dibenzocyclooctadiene (Whiting, 1985) and dibenzylbutyrolactol (Figure 1). Lignans of each subgroup vary substantially in oxidation levels of both the aromatic rings and propyl side chains. Some lignans of furan, dibenzylbutane, and dibenzocyclooctadiene have no oxygen at C9 (C9 ) (Figures 1 and 2), while some lignans have extra hydroxyl groups at C7 (C7 ) or C8 (C8 ) (e.g. podophyllotoxin 40 with C7 OH and wikstromol 33 with C8OH, Figure 2). 3-Methoxy-4-hydroxyphenyl (guaiacyl), 3,4-dimethoxyphenyl (veratryl), 3,4methylenedioxyphenyl (piperonyl), 3,5-dimethoxy-4hydroxyphenyl (syringyl), and 3,4,5-trimethoxyphenyl (Figure 1) are the most frequently occurring aromatic rings found in lignans. 4-Hydroxyphenyl and 3,4-dihydroxyphenyl lignans have also been identied (Ayres and Loike, 1990), though they are rare.

Enantiomeric diversity of lignans In addition to the structural diversity, lignans vary substantially with respect to enantiomeric composition (Umezawa et al., 1997a). First of all, it is important to compare the absolute congurations of typical lignans. The following lignans have the same absolute congurations at C8 and C8 with respect to the carbon skeleton (Figures 2 and 3) (Umezawa et al., 1997a; Umezawa, 2001): (+)-pinoresinol (+)-1 and syringaresinol (+)-3 (furofuran); (+)-lariciresinol (+)-18 (furan);

Table 1. Enantiomeric compositions of lignans isolated from various plant species Lignans and their enantiomeric compositions (% e.e.) Furofuran Furan Dibenzylbutane Dibenzylbutyrolactone (-)-MR (>99% e.e.) (-)-AR (>99% e.e.) (-)-SIR (65% e.e.)

Plant classication rank Family Species Arctium lappa (seed) Arctium lappa (petiole) Forsythia koreana (+)-PR (82% e.e.) (+)-LR (35% e.e.) (-)-SIR (>99% e.e.) (+)-SIR (81% e.e.) (-)-MR (>99% e.e.) (-)-AR (>99% e.e.) (-)-MR (>99% e.e.) (-)-AR (>99% e.e.)

Reference Umezawa and Shimada, 1996b Suzuki et al., 1998 Suzuki et al., 2002a

Asteraceae

Oleaceae

(-)-SIR (>99% e.e.) (+)-PR (65% e.e.) (+)-LR (70% e.e.) (-)-SIR (>99% e.e.) (+)-SIR (98% e.e.)

Linaceae

Umezawa et al., 1992 Umezawa et al., 1994 Umezawa et al., Unpublished Umezawa et al., 1991 Ozawa et al., 1993 Mikame et al., 2002 Umezawa et al., 1997b

Euphorbiaceae

Forsythia intermedia Linum avum var. compactum Phyllanthus sp.

373

374

Table 1. Continued Lignans and their enantiomeric compositions (% e.e.) Furofuran Furan Dibenzylbutane Dibenzylbutyrolactone (-)-PR (74% e.e.) (-)-LR (39% e.e.) (-)-SIR (45% e.e.)

Plant classication rank Family Species Wikstroemia sikokiana

Reference Umezawa and Shimada, 1996a Okunishi et al., 2000

Thymelaeaceae

Pinaceae Cupressaceae

Daphne odora Daphne genkwa Larix leptolepis Chamaecyparis obtusa (cv. Breviramea) Thuja occidentalis

(-)-PR (95% e.e.) (-)-PR (88% e.e.) (+)-PR (92% e.e.)

(-)-LR (89% e.e.) (-)-LR (88% e.e.)

(+)-SIR (>99% e.e.) (+)-SIR (97% e.e.)

(+)-MR (>99% e.e.) (+)-KU (>99% e.e.) (+)-WI (>99% e.e.) (+)-MeTR (>99% e.e.) (+)-MR (>99% e.e.) (+)-MR (>99% e.e.)

Okunishi et al., 2001 Okunishi et al., 2001 Nabeta et al., 1991 Takaku et al., 2001

(-)-MR (>99% e.e.) (-)-HI (>99% e.e.) (-)-HA (>99% e.e.) (-)-PL (>99% e.e.) (-)-MR (>99% e.e.) (-)-TJM (>99% e.e.) (-)-DMYA (>99% e.e.) (-)-WI (>99% e.e.)

Kawai et al., 1999

PR, pinoresinol 1; LR, lariciresinol 18; SIR, secoisolariciresinol 23; MR, matairesinol 27; AR, arctigenin 28; HI, hinokinin 29; PL, pluviatolide 30; WI, wikstromol 33;

KU, kusunokinin 67; HA, haplomyrfolin 68; MeTR, methyltrachelogenin 69; TJM, thujaplicatin methyl ether 70; DMYA, 4-demethylyatein 71

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Figure 1. Basic skeletons of lignans.

iciresinol differ among various plant species (Umezawa et al., 1997a). In contrast, most dibenzylbutyrolactone lignans are levorotatory (Umezawa et al., 1997a), except that the lignans of this subgroup isolated from Thymelaeaceae plants such as Wikstroemia spp., Daphne spp., and Stellera chamaejasme (Umezawa and Shimada, 1996a; Umezawa et al., 1997a; Okunishi et al., 2000, 2001; Chen et al., 2001; Xu et al., 2001) (Table 1) and Selaginella doederleinii (Selaginellaceae) (Lin et al., 1994) are dextrorotatory. Furthermore, within a single plant species the absolute congurations of predominant lignan enantiomers may vary (Table 1) (Umezawa et al., 1997a). For instance, (+)-arctigenin (+)-28 (Suzuki et al., 1982) and (+)-pinoresinol (+)-1 (Tandon and Rastogi, 1976) isolated from Wikstroemia indica (= W. viridiora) have opposite absolute congurations to each other at C8 and C8 . Similarly, W . sikokiana produces (+)-matairesinol (+)-27 (optically pure) (Umezawa and Shimada, 1996a) and (-)-secoisolariciresinol (-)-23 (45% e.e.) (Okunishi et al., 2000), and the predominant enantiomers of the lignans have opposite absolute congurations (Table 1). In addition, it is noteworthy that the predominant enantiomer of secoisolariciresinol 23 isolated from A. lappa seeds is opposite to that from A. lappa petioles; (-)-23 (65% e.e.) was obtained from the seeds and (+)-23 (81% e.e.) was from the petioles (Umezawa and Shimada,

1996b; Suzuki et al., 1998, 2002a) (Table 1). In contrast, the following lignans from Forsythia spp. have the same absolute congurations at C8 and C8 : (+)-pinoresinol (+)-1 (Kitagawa et al., 1988; Umezawa et al., 1990b, 1992), (+)-lariciresinol (+)-18 (Umezawa et al., 1994), (-)-secoisolariciresinol (-)-23 (Umezawa et al., 1991, 1992), (-)-matairesinol (-)-27 (Nishibe et al., 1988; Kitagawa et al., 1988; Rahman et al., 1990; Umezawa et al., 1991, 1992) and (-)arctigenin (-)-28 (Nishibe et al., 1988; Kitagawa et al., 1988; Rahman et al., 1990; Umezawa et al., 1992; Ozawa et al., 1993). The enantiomeric properties of lignans can be described with four statements. First, dibenzylbutyrolactone lignans are optically pure (>99% e.e.), while furofuran and furan lignans are mixtures of both enantiomers and exhibit various enantiomeric compositions. Second, dibenzylbutyrolactone lignans are for the most part levorotatory except that the lignans from Thymelaeaceae plants and S. doederleinii are dextrorotatory. Third, predominant enantiomers of furofuran, furan, and dibenzylbutane lignans vary among plant species, and even within different organs in a single plant species (A. lappa). Fourth, the absolute congurations of the predominant enantiomers of various lignans isolated from a single plant species are sometimes different.

376

Figure 2. Lignans obtained in the CAPLUS database search. Note that only one enantiomer of each lignan is shown.

377

Figure 2. Continued

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Figure 2. Continued

Biosynthetic pathways of lignans The biosynthetic pathway of several of the typical lignans listed in Figure 2 have been well established. However, the biosynthetic pathway for many other lignans still remains unknown (Scheme 1). Erdtman (1933) rst suggested that the basic lignan structure was formed by the coupling of two phenylpropanoid monomer units. Later, this assumption was substantiated by isotope tracer experiments. These investigations were outlined in a number of reviews, for example, by Dewick (1989), Ayres and Loike (1990), and Umezawa (1997). In 1990 the rst example of in vitro formation of optically pure (-)-secoisolariciresinol (-)-23 from an achiral phenylpropanoid monomer, coniferyl alcohol, was reported using Forsythia intermedia as an enzyme source (Umezawa et al., 1990a). At the same time, selective oxidation of (-)-secoisolariciresinol (-)-23 to (-)-matairesinol (-)-27 by an enzyme preparation from F. intermedia was reported (Umezawa et al., 1990b, 1991). Later, this enzyme, secoisolariciresinol dehydrogenase (SIRD), was puried and its cDNA was cloned (Xia et al., 2001). The selective reduction of (+)-enantiomer of pinoresinol (+)-1 to give rise to (-)-

secoisolariciresinol (-)-23 via (+)-lariciresinol (+)-18 by Forsythia enzyme was also detected (Katayama et al., 1992, 1993; Umezawa et al., 1994). This enzyme, pinoresinol/lariciresinol reductase (PLR), was puried and its cDNA was cloned (Chu et al., 1993; Dinkova-Kostova et al., 1996). Davin et al. (1997) isolated a unique protein from Forsythia sp. that led to enantioselective formation of (+)-pinoresinol (+)1 by coupling of coniferyl alcohol in the presence of laccase/O2 . The function of the unique protein [dirigent protein (DP)] as an asymmetric inducer is very important and interesting from a stereochemical aspect. These studies demonstrated that lignan synthesis mediated by Forsythia DP and enzymes is well controlled in terms of stereochemistry. Studies in a wide range of gymnosperms and angiosperms showed the existence of a common lignan biosynthetic pathway. PLR and SIRD have been detected from several plant species (Xia et al., 2000; Umezawa and Shimada, 1996b; Katayama et al., 1997; Suzuki et al., 1998, 2002b, 2002c; Okunishi et al., 2001). In addition, administration of [2 H] or [14 C]coniferyl alcohol to a variety of plant species resulted in the incorporation of 2 H or 14 C into pinores-

Scheme 1. Possible biosynthetic pathways for various types of lignans. Solid and broken arrows represent pathways substantiated by experiments and assumed based on comparison of chemical structures, respectively.

379

380 inol 1, lariciresinol 18, secoisolariciresinol 23, and/or matairesinol 27 (Umezawa et al., 1997b, 1998; Miyauchi and Ozawa, 1998; Kato et al., 1998; Kawai et al., 1999; Takaku et al., 2001). These plants represent a wide range of angiosperms and gymnosperms. Therefore the conversion, coniferyl alcohol pinoresinol 1 (furofuran) lariciresinol 18 (furan) secoisolariciresinol 23 (dibenzylbutane) matairesinol 27 (dibenzylbutyrolactone) (Scheme 1), appears to be a common lignan biosynthetic pathway. On the other hand, in terms of stereochemistry, the conversion from coniferyl alcohol to matairesinol 27 involves great diversity. Thus, as mentioned, the farthest upstream lignans in the biosynthetic pathway, pinoresinol 1, lariciresinol 18, and secoisolariciresinol 23, isolated from various plant species, were composed of both enantiomers (with various enantiomeric compositions), and were not optically pure (Table 1) (Umezawa et al., 1997a; Umezawa, 2001). In sharp contrast, all dibenzylbutyrolactone lignans, including matairesinol 27, for which enantiomeric compositions have been determined using chiral HPLC, were optically pure regardless of the sign of optical rotation (Table 1) (Umezawa et al., 1997a; Umezawa, 2001). These facts unequivocally indicated that not only was the entrance step mediated by DP involved, but subsequent metabolic steps catalyzed by PLR and SIRD were also involved in determining the enantiomeric composition of lignans or the production of optically pure lignans (Umezawa, 2001). It was also suggested that the variation of enantiomeric compositions of the upstream lignans (lariciresinol 18, secoisolariciresinol 23, and matairesinol 27) among different plant species may be ascribed, at least in part, to the characteristics of the reactions catalyzed by PLRs and SIRDs as well as their expression patterns (Umezawa, 2001). This view was supported at least in part by enzymatic experiments as follows. As mentioned (Table 1), different enantiomers of secoisolariciresinol 23 occur predominantly in different organs of Arctium lappa; (-)-secoisolariciresinol (-)-23 (65% e.e.) was isolated from the seeds, while (+)-enantiomer (+)-23 (81% e.e.) was isolated from the petioles. The enantiomeric segregation was also demonstrated by in vitro experiments with enzyme preparations from A. lappa seeds and petioles. The petiole enzyme preparation catalyzed the formation of (+)-secoisolariciresinol (+)-23 (20% e.e.) from achiral coniferyl alcohol in the presence of NADPH and H2 O2 , whereas that from ripening seeds catalyzed the formation of (-)-secoisolariciresinol (-)-23 (38% e.e.) under the same conditions. Because the assay with only H2 O2 as a cofactor exhibited signicant pinoresinol formation, PLR-catalyzed reduction of once-formed pinoresinol 1 probably resulted in secoisolariciresinol formation. In addition, incubation of ()-pinoresinols ()-1 and ()-lariciresinols ()-18 with a PRL preparation from the seeds gave (-)-secoisolariciresinol (-)-23 with high enantiomer excess (99 and 91% e.e., respectively), whereas (-)-secoisolariciresinol (-)-23 with rather low enantiomer excess (44 and 37% e.e.) was obtained following incubation of ()-pinoresinols ()-1 and ()lariciresinols ()-18, respectively, with a PRL preparation from the petioles (Umezawa and Shimada, 1996b; Suzuki et al., 1998; Suzuki et al., 1998, 2002b). The stereochemical results were accounted for by postulating that A. lappa has PLR isoforms showing different selectivity in terms of enantiomers of the substrates and that the isoforms are expressed differentially in A. lappa organs (Suzuki et al., 2002b). Although nal conclusions await further experiments, this view is in line with recent ndings on PLR from various plant species. PLRs from F . intermedia (Chu et al., 1993; Dinkova-Kostova et al., 1996) and Zanthoxylum ailanthoides (Katayama et al., 1997) catalyzed the selective formation of (+)-lariciresinol (+)-18 and (-)-secoisolariciresinol (-)-23 from ()pinoresinols ()-1, whereas that from Daphne genkwa catalyzed the selective formation of (-)-lariciresinol (-)-18 from ()-pinoresinols ()-1 (Okunishi et al., 2001). Furthermore, the presence of cDNAs corresponding to the two stereochemically distinct PLRs was demonstrated in a single plant species (Thuja plicata) (Fujita et al., 1999). As for SIRD, several papers dealing with the stereochemistry of SIRD-catalyzed reactions have been published. SIRDs of F . intermedia (Umezawa et al., 1990b, 1991; Xia et al., 2001) and A. lappa (Suzuki et al., 2002b) catalyzed the selective formation of (-)-matairesinol (-)-27 from ()-secoisolariciresinols ()-23. Considering the consistency of the enantiomers between the naturally occurring and in vitro formed matairesinols 27 in the cases of Forsythia and Arctium, it seemed likely that (+)-matairesinol forming SIRD would be detected in (+)-matairesinol producing Thymelaeaceae plants. However, it has been found that this is not the case; D. genkwa and Daphne odora SIRDs catalyzed selective formation of (-)-matairesinol (-)-27 from ()-secoisolariciresinols ()-23 (Okunishi et al., 2004). Thus, mechanisms for the (+)-matairesinol formation remain unclear and the

381

Figure 3. Some of the dibenzylbutyrolactone lignans which appear in Table 1. The other dibenzylbutyrolactone lignans are shown in Figure 2.

results present a new question about the stereochemistry of lignan biosynthesis. It was established that the conversion of secoisolariciresinol 23 to matairesinol 27 proceeded via the intermediary dibenzylbutyrolactollignan 79 using recombinant F . intermedia and Podophyllum peltatum SIRDs. This result provides insight into the biosynthesis of dibenzylbutyrolactols. For example, dibenzylbutyrolactollignan 79 occurring in Abies pinsapo (Barrero et al., 1994) and Cedrus deodara (Tiwari et al., 2001) may be formed from secoisolariciresinol 23 by SIRD or dehydrogenases similar to SIRDs (Scheme 1). Biosynthetic pathways for many other lignans can be regarded as starting from the upstream lignans, pinoresinol 1, lariciresinol 18, secoisolariciresinol 23, and matairesinol 27 (Scheme 1). Among them, the pathway toward an important antitumor lignan, podophyllotoxin 40, has been substantiated by tracer and enzymatic experiments. In the 1980s Dewick and his colleagues conducted a series of detailed feeding experiments with radio-labelled precursors and revealed the pathways from yatein 31 to podophyllotoxin 40 via deoxypodophyllotoxin (= desoxypodophyllotoxin, anthricin) 45 (Jackson and Dewick, 1984a, b; Kamil and Dewick, 1986a, b; Dewick, 1989). Next, they showed that matairesinol 27 was metabolized to podophyllotoxin 40 and proposed yatein 31 as a possible intermediate between matairesinol 27 and podophyllotoxin 40 (Broomhead et al., 1991). The transformation of matairesinol 27 to yatein 31 involves four steps: 5-hydroxylation, dual methylation at C4OH and C5OH, and methylenedioxy bridge formation at C3 and C4. Many possible sequences of these four steps can be envisaged. Recently based on metabolic proling and a series of stable isotope tracer experiments using Anthriscus sylvestris (Scheme 2), Sakakibara et al. (2003) demonstrated a direct pathway and not a metabolic grid from matairesinol 27

to yatein 31 via thujaplicatin 80. It was also demonstrated that the pathway was accompanied by a branch from matairesinol 27 to bursehernin 83 which did not lead to yatein 31 (Scheme 2) (Sakakibara et al., 2003). The transformation of yatein 31 to deoxypodophyllotoxin 45 has not yet been detected. But, several enzymes involved in the further transformation of deoxypodophyllotoxin 45 to podophyllotoxin 40 (Scheme 1), -peltatin 42, and methoxypodophyllotoxin have been reported (Petersen and Alfermann, 2001; Molog et al., 2001; Kuhlmann et al., 2002). Taken together, these data provide an outline of the biosynthetic pathways which may exist for several aryltetralin lactone lignans. Enzymes involved in the formation of the 3,4-dimethoxyphenyl and 3,4-methylenedioxyphenyl groups which are present in lignans (Figure 2) have been reported. Ozawa et al. detected Omethyltransferase activities converting pinoresinol 1 and matairesinol 27 to eudesmin 4 and arctigenin 28, respectively (Ozawa et al., 1993; Miyauchi and Ozawa, 1998). A P-450 monooxygenase obtained from the microsomal fraction of Sesamun indicum seeds catalyzed the conversion of pinoresinol 1 to piperitol (mono 3,4-methylenedioxyphenyl analogue of pinoresinol) (Jiao et al., 1998). Some lignans have hydroxyl groups at C7 (C7 ) or C8 (C8 ) of their side chain [e.g. wikstromol 33, trachelogenin 34, olivil 21, and podophyllotoxin 40 (Figure 2)]. The 7 (7 )- or 8 (8 )-hydroxylated lignans are probably formed from the corresponding deoxy analogs. Recently, 7 -hydroxylation of matairesinol 27 (Xia et al., 2000) and deoxypodophyllotoxin 45 (Petersen and Alfermann, 2001; Kuhlmann et al., 2002) were reported. As for biosynthesis of arylnaphthalenes, Broomhead et al. (1991) administered 14 C-labeled matairesinol 27 to Diphylleia cymosa, but no incorporation of 14 C into an arylnaphthalene lignan diphyllin 52

382

Scheme 2. Biosynthetic pathways for yatein and bursehernin in Anthriscus sylvestris.

was detected. Hence, only speculative discussion based on a comparison of their chemical structures with lignans whose biosynthetic pathways are known is possible. Considering the structural similarity of arylnaphthalenes to aryltetralins, it seems likely that arylnaphthalenes are formed by dehydrogenation of the corresponding aryltetralins or by hydroxylation of aryltetralins followed by dehydration (Scheme 1). Some aryltetralin lignans have two OH groups at C9 and C9 (Figure 2). The 9,9 -dihydroxyaryltetralin lignans (e.g. isolariciresinol 36 and lyoniresinol 37) may be formed by a rearrangement of the corresponding furan lignans (lariciresinol 18 and 5,5dimethoxylaricirsinol, respectively) or by oxidation of the corresponding dibenzylbutane lignans followed by cyclization (Scheme 1). Dibenzocyclooctadiene lignans can be divided into two categories based on their oxidation states at C9 and C9 . One category includes dibenzocyclooctadiene lignans absent oxygen at C9 (C9 ) (e.g. gomisin A 62 and wuweizisu C 66, Figure 2). The other category is butyrolactone, steganacin 54 (Figure 2). They might be formed from the corresponding seco analogs without the biphenyl bond (Scheme 1). Plants producing dibenzocyclooctadiene lignans without 9(9 )-oxygen are restricted to Illiciales (Magnoliidae) (Table 2), while many dibenzylbutane and furan lignans without oxygen at C9 and C9 were isolated especially from Magnoliidae plants. It is noteworthy that some plants of Magnoliidae are also good sources of allyl- and propenylphenols such as eugenol, safrole, and anethole which have no oxygen at C9 (Gibbs, 1974). This suggests that lignans without 9(9 )-oxygen may be formed by a coupling of propenylphenols such as isoeugenol (Scheme1), as proposed by Gottlieb (1972). Recently, Moinuddin et al. (2003) proposed coupling of p-anol (demeth-

oxyisoeugenol) to give several furan lignans without 9(9 )-oxygen in Larrea tridentata (Zygophyllaceae). During the last decade, lignans of another type have been isolated from liverworts (Hepaticopsida). An optically active dicarboxylic acid lignan, epiphyllic acid 72 (a caffeic acid dimer, Figure 4), and its derivatives, that do not belong to the 12 subgroups of typical lignans shown in Figure 2, were isolated from several liverworts (Cullmann et al., 1993, 1996, 1999; Martini et al., 1998; Cullmann and Becker, 1999; Tazaki, 2000; Tazaki et al., 2002; Scher et al., 2003). Dicarboxylic acid lignans (p-hydroxycinnamate dimers) and their esters and amides also occur in some vascular plants: thomasidioic acid 73 (Figure 4) isolated from Ulmus thomasii (Ulmaceae) (Hostettler and Seikel, 1969), rabdosiin 74 (a rosmarinic acid dimer, Figure 4) from Rabdosia japonica (Lamiaceae) (Agata et al., 1988) and Lithospermum erythrorhizon (Boraginaceae) (Yamamoto et al., 2000), arillatose A (a sucrose ester of thomasidioic acid 73) from Polygala arillata (Polygalaceae) (Kobayashi et al., 2000), chilianthin A (= rhoipteleic acid A, an triterpene ester of epiphyllic acid 72) from Rhoiptelea chiliantha (Rhoipteleaceae) (Jiang et al., 1996), and cannabisins B, C, and D 75, 76, and 77 (Figure 4) from Cannabis sativa (Cannabaceae) (Sakakibara et al., 1992). Tazaki et al. (1999) demonstrated that caffeic acid was converted to epiphyllic acid 72 in a liverwort Lophocolea heterophylla (Geocalycaceae) (Scheme 1), while nothing is known about the biosynthesis of this type of lignans in vascular plants. Phylogenetic distribution of lignans Recent developments in molecular biology provide useful tools for analyzing the molecular evolution of proteins. However, he numbers of cloned cDNAs

383

Figure 4. Examples of dicarboxylic acid lignans (p-hydroxycinnamate dimers).

involved in lignan biosynthesis are still too limited to conduct a comprehensive and systematic analysis of the molecular evolution of lignan biosynthesis. Additionally, fossil records do not provide information about the appearance of phenylpropanoid biosynthesis. Consequently, in this review the author correlates the accumulation of lignans to extant plant species. First, 308 typical lignans listed by Ayres and Loike (1990) were subjected to a database search irregardless of their signs of optical rotation [SciFinder Search; database, Chemical Abstracts Plus (CAPLUS); keywords, the name of each lignan (e.g. pinoresinol) and isolation. Lignans which appeared in more than 10 papers were chosen. This gave the 66 lignans, shown in Figure 2. Lignan glycosides (e.g. arctiin) and acylated lignans (e.g. acetoxypinoresinol) were treated as the corresponding aglycones (e.g. arctigenin 28) and deacylated lignans (e.g. hydroxypinoresinol 9), respectively, except for steganacin 54 and gomisin B 64. As shown in Figure 2, the selected lignans were classied into 12 subgroups based upon their possible biosynthetic pathways (Scheme 1): furofuran; furan with 9(9 )-oxygen; furan without 9(9 )-oxygen; 9,9 -dihydoxydibenzylbutane; dibenzylbutane without 9(9 )-oxygen; dibenzylbutyrolactol; dibenzylbutyrolactone; 9,9 -dihydroxyaryltetralin; aryltetralin lactone; arylnaphthalene; dibenzocyclooctadiene lactone; and dibenzocyclooctadiene without 9(9 )-oxygen. Next, plant species producing at least one of the 66 lignans were arranged phylogenetically (Table 2). The plant classication for Magnoliophyta is due to Cronquist (1981) and Kato (1997) and for Gymnospermophyta and Pteridophyta it is Kramer and Green (1990) and Kato (1997). Due to the limited length of this paper, only plant families of the lignan-producing plant species versus the lignan subgroups are listed. The detailed phylogenetic list of lignan producing plant species in relation to the 66 lignans which com-

plements Table 2 is available elsewhere (Umezawa, 2003). As mentioned, the dicarboxylic acid lignans (phydroxycinnamate dimers) isolated recently from liverworts and some Magnoliopsida plants. They were not obtained in the present database search, but this is mainly because many of them have been isolated after the publication of the book by Ayres and Loike (1990). Hence, the dicarboxylic acid lignans are also included in Table 2. The classication of Bryophyta is due to Furuki and Mizutani (1994). Table 2 shows that Coniferopsida are especially well represented. Lignan producing plants are distributed throughout the six subclasses of Magnoliopsida (Table 2). In contrast, Liliopsida plants are rather poor lignan sources; only eight families of the class contained lignan producing plant species (Table 2). Most of them are furofuran lignans which are upstream metabolites in the biosynthetic pathways (Scheme 1). Three examples of other subgroups [secoisolariciresinol 23 (dibenzylbutane) from Hymenocallis littoralis (Liliaceae) (Lin et al., 1995), lariciresinol 18 and its glucosides (furan) from Arum italicum (Araceae) (Della Greca et al., 1993), and grandisin 58 (furan) from Rhaphidophora decursiva (Araceae) (Zhang et al., 2001)] were obtained in the present database search (Table 2). There were 108 families of lignan producing plants in the division Magnoliophyta (Table 2), which was twice that (51 families) reported by MacRae and Towers about 20 years ago (MacRae and Towers, 1984). The increase is probably due to the accumulation of phytochemical data during the last two decades. It is noteworthy that Table 2 is in line with that presented by MacRae and Towers (1984), i.e. Magnoliidae, Rosidae, and Asteridae include many lignan producing families compared with the total family numbers of each subclass. The phylogenetic distribution of lignan producing plant species obtained in the present data base search (Table 2) shows several important characteristics in

384

Table 2. Distribution of lignans in plant kingdom.

Table 2. (Continued.)

385

386

Table 2. (Continued.)

Solid box represents the occurrence of lignans of each subgroup in respective plant families.

387 relation to lignan biosynthesis. First, the occurrence of lignans without 9(9 )-oxygen (furan, dibenzylbutane, and dibenzocyclooctadiene) is rather restricted. They are found in one order (Sapindales) of Rosidae, six orders of Magnoliidae, and one order (Arales) of Arecidae. Interestingly, this coincides with the fact that some Magnoliidae plants are good sources of phenylpropanoid monomers with propenyl or allyl side chains (e.g., isoeugenol, safrole, and anethole) (Gibbs, 1974), suggesting that the evolution of the biosynthesis of lignans without 9(9 )-oxygen is closely related to that of other phenylpropanoids without oxygen at C9. Second, furofurans are the most widely distributed lignans (Table 2). In Coniferales plants, whose lignins are predominantly the guaiacyl type, pinoresinol 1 with two guaiacyl aromatic rings occurs widely, whereas neither medioresinol 2 nor syringaresinol 3, both having at least one 3,5-dimethoxy4-hydroxyphenyl (syringyl) group), were detected (Umezawa, 2003). In contrast, there are many examples of the simultaneous occurrence of pinoresinol 1 (or furofuran lignans with two 3,4-dialkoxyphenyl groups) and syringaresinol 3 (or furofuran lignans with one or two 3,4,5-trialkoxyphenyl groups) in a single species of angiosperm (Magnoliopsida) which produce both guaiacyl and syringyl lignins (Umezawa, 2003). These facts, together with the above-mentioned case of lignans without 9(9 )-oxygen, suggest a close relationship between the evolution of lignan biosynthesis and that of other phenylpropanoid compounds including lignins and phenylpropanoid monomers. Third, in contrast, there is evidence to suggest that a lignan biosynthetic system was acquired prior to that of a lignin biosynthetic system. In addition to the dicarboxylic acid lignans (p-hydroxycinnamate dimers) (Table 2), liverworts produce other phenylpropanoid compounds such as avonoids (Zinsmeister et al., 1991). Lignins, however, have not been detected in liverworts (Erickson and Miksche, 1974; Miksche and Yasuda, 1978), indicating that the emergence of epiphyllic acid biosynthesis occurred prior to that of lignin biosynthesis. In addition, the typical lignans derived from coniferyl alcohol, such as pinoresinol 1, lariciresinol 18, secoisolariciresinol 23, and matairesinol 27, have not yet been isolated from liverworts (Table 2). These facts strongly suggest that liverworts lack the reduction pathway from p-hydroxycinnamic acids to p-hydroxycinnamyl alcohols, and, therefore lack the biosynthetic systems for lignans and lignins involving p-hydroxycinnamyl alcohols as intermediates. The point of emergence and evolution of the biosynthetic systems for p-hydroxycinnamyl alcohols and the compounds derived thereof are of special interest in connection with the evolution of vascular plants. Also, it is of interest to elucidate the evolutionary relationship between the biosynthesis of epiphyllic acid 72 in liverworts and that of the dicarboxylic acid lignans in vascular plants. Fourth, distribution of dextrorotatory dibenzylbutyrolactone lignans is also of special interest. As mentioned, most dibenzylbutyrolactone lignans are levorotatory and have the same absolute conguration at C8 and C8 in terms of the carbon skeleton, whereas the lignans occurring in Thymelaeaceae plants (Table 1) (Umezawa et al., 1997a; Umezawa, 2001) and Selaginella doederleinii (Lin et al., 1994) are dextrorotatory. Thymelaeaceae and Selaginella plants belong to the divisions Magnoliophyta and Pteridophyta, respectively, and both divisions are phylogenetically far apart. This spotty distribution of dextrorotatory dibenzylbutyrolactone lignans suggests a parallel evolution of some lignan biosynthetic systems in phylogenetically distant taxa.

Conclusions Lignans show considerable diversity in terms of basic structure. Typical lignans can be classied into 12 subgroups based on their basic structures and oxidation levels at C9 (C9 ). Lignans exist in diverse enantiomeric compositions. Dibenzylbutyrolactone lignans, which occur late in the biosynthetic pathway, are optically pure. On the other hand, the most biosynthetically upstream lignans, furofuran and furan, are mixtures of both enantiomers and exhibit various enantiomeric compositions. This indicates that lignan enantiomeric compositions are determined through several upstream reaction steps mediated by dirigent protein (DP), pinoresinol/lariciresinol reductase (PLR), and secoisolariciresinol dehydrogenase (SIRD). In addition, most dibenzylbutyrolactone lignans are levorotatory, whereas those from Thymelaeaceae plants and Selaginella doederleinii (Selaginellaceae) are dextrorotatory. The spotty distribution of dextrorotatory dibenzylbutyrolactone lignans among vascular plants suggests a parallel evolution of the biosynthetic systems for the dextrorotatory dibenzylbutyrolactone lignans.

388 It was also suggested that the acquisition and evolution of the biosynthesis of a variety of lignans in vascular plants may occur in relation to those of lignins and other related phenylpropanoid monomers, while the lignan biosynthetic system in liverworts probably appeared prior to lignin biosynthesis.
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Acknowledgements The author thanks Dr Hiroyuki Kuroda, Wood Research Institute, Kyoto University, for his suggestion on chemotaxonomy. Thanks are also due to Dr Tatsuwo Furuki, Natural History Museum & Institute, Chiba, for his suggestion on the classication of liverworts, and Dr Hiroyuki Tazaki, Obihiro University of Agriculture and Veterinary Medicine, for the information on liverwort lignans. The author is indebted to Jacqueline Leshkevich for valuable comments on English writing.

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