Current understanding of lignan biosynthesis

Lignans constitute a large and diverse class of molecules widely distributed throughout the plant kingdom. They exhibit a wide range of pharmaceutically relevant biological activities and have attracted widespread research interests. This review covers the current understanding of lignan biosynthesis and aims to highlight key biosynthetic transformations responsible for their structural and biological diversity


Introduction
Lignans constitute a large and diverse class of natural products widely distributed in the plant kingdom and are found in plant roots, rhizomes, stems, leaves, seeds and fruits. 1 They are derived from the stereoselective oxidative coupling of two phenylpropanoid monomers, also termed monolignols, at the olefin of the propenyl moiety.The resulting β-β' (C8-C8') bond defines the substance class.Another defining characteristic of lignans is that oxygen represents the only heteroatom incorporated into the scaffold.Despite this rather narrow definition and common origin, lignans exhibit a vast structural diversity.A highly divergent biosynthesis leads to different patterns in cyclization, dividing lignans into structural subtypes: furofurans, furans, dibenzylbutanes, dibenzyllactones, dibenzylcyclooctadienes, aryltetralins and arylnaphthalenes. 2 An overview of the structural features, nomenclature and classification is provided in Figure 1.
4][5][6] While their specific functions in planta are largely unknown, lignans are thought to be involved in processes related to plant physiology and development.Furthermore, lignans are implicated to serve important functions in plant defense and survival against ecological stressors, as evidenced by their bioactive properties and distribution across numerous plant tissues.Many lignan-rich plants have been known for their therapeutic properties and potential health benefits to humans and have a long history of use in traditional medicine and diet. 7Prominent examples include flax (Linum usitatissimum), sesame (Sesamum indicum), forsythia (Forsythia suspensa), olive (Olea europaea) and schisandra (Schisandra chinensis).
One of the most well-known lignans is podophyllotoxin from mayapple species (Podophyllum peltatum, Sinopodophyllum hexandrum).Podophyllotoxin is a potent anti-cancer and anti-viral agent that prevents cell division by destabilizing microtubules.It is a FDA-approved drug used for the treatment of external warts cause by human papillomavirus (HPV) since 1990, while it has been used since 1942. 8Medicinal use of Podophyllum was reported as early as 900 AD in The Leech Book of Bald, an English pharmaceutical reference book. 9Since the 1970s, dozens of podophyllotoxin derived drugs have entered clinical trials.Deoxypodophyllotoxin, a biosynthetic precursor to podophyllotoxin, was approved for phase I trials by the National Medical Products Administration in China in 2017. 10Its semi-synthetic glycosylated congeners etoposide and teniposide were © AUTHOR(S) approved for medical use in the United States in 1983 and 1992 respectively.Both of them are used for the treatment of several cancer types, including lung cancer, lymphoma, leukemia, neuroblastoma and testicular cancer.][13] Nordihydroguaiaretic acid (NDGA) has emerged as another promising lignan lead compound.It was isolated from the creosote bush (Larrea tridentata), which has been used in traditional medicine in northern Mexico and the southwestern United States for the treatment of more than 50 diseases. 14It was shown to exhibit potent antioxidant, antiviral and antineoplastic activities.NDGA was used as a food antioxidant in the United States until the 1970s.Since then, it has been approved for treatment of actinic keratosis and its methylated derivative tetra-O-methyl NDGA has entered clinical trials for the treatment of HPV-linked cervical intraepithelial neoplasia.Both compounds have been identified to inhibit replication of dengue virus, hepatitis C virus, West Nile Virus and Zika Virus in vitro. 15,16More than 150 dibenzylcyclooctadiene lignans, also termed Schisandra chinensis lignans, have been characterized to date and were all isolated from species in the Schisandraceae family. 17Dibenzylcyclooctadienes make up the largest lignan subclass, however little is known about their biosynthesis.Schisandra chinensis has a long history of use in traditional Chinese medicine for the treatment of hepatitis and myocardial disorders among others.Notably, schizandrin A and B have been investigated for their antiviral activity against HIV, and schizandrin C was shown to possess potent hepatoprotectant properties. 18,19chizandrin C derivative was approved for the treatment of hepatotoxin-induced liver injury by the Chinese Food and Drug Administration in 2004. 20Sesamin has been shown to exhibit potent hyaluronidase inhibition activity, thus attracting interest from the cosmetic industry. 21It is the major lignan found in sesame seeds, a historical staple food in Africa and Asia and it has been popularized worldwide.Investigations about its health benefits have demonstrated anti-inflammatory and antioxidant properties. 22,23Structures of several representative lignan natural products are shown in Figure 2. A comprehensive summary of bioactivities of lignan natural products can be found in recent reviews by Osmakov et al. 24  Unsurprisingly, the sheer number (more than 750 lignans have been discovered since 2016) and diversity, along with substantial therapeutic potential have made lignan natural products a rich source of lead compounds for drug development. 26However, natural occurrence in the producing organisms is often very low.For example, podophyllotoxin accounts for 0.3-1.0%(w/w) of rhizome mass, while sesamin constitutes 0.4-0.6%(w/w) of sesame oil, making extraction laborious and uneconomical. 27,28An estimated 300,000 lbs. of mayapple roots are harvested per year to cover the demand of podophyllotoxin and epipodophyllotoxin, the precursor to etoposide and teniposide. 29As mayapple species are not suitable to farm, wild S. hexandrum has become an endangered species. 30,317 An ever-growing need for effective disease treatment and a recent advent of renewed interest in natural medicines and cosmetics have led to an increased global demand for lignan lead compounds.The lignan market is estimated to reach over 610 million USD by 2028. 38It is therefore imperative to gain insight into the biosynthesis of lignan to aid in the identification of genes and elucidation of the corresponding enzyme mechanisms, providing a foundation for developing sustainable and economical methods of production.Nature effortlessly and efficiently accomplishes enormous structural diversity using a comparatively limited set of © AUTHOR(S) enzymatic transformations.In this review, we provide an outline of a concerted pathway that is deployed to generate the scaffolds of lignans, which become branchpoints for the production of diverse natural products.

Formation of Coniferyl Alcohol
Before diving into the intricacies of lignan biosynthesis, it is important to discuss the biogenesis of their common precursor, coniferyl alcohol (CA) (Figure 3).In phenylpropanoid metabolism, as is the case for many other secondary metabolite pathways, amino acids, diverted from protein synthesis act as an entry point from which the natural product scaffold is built upon.For lignans, as well as flavanones, stilbenes, chalcones and other natural product families originating from the phenylpropanoid pathway, phenylalanine and tyrosine represent this entry point. 39,40Phenylalanine ammonia-lyase (PAL) catalyzes elimination of ammonia to generate the cinnamic acid which is subsequently hydroxylated in the C4-position by cytochrome P450 monooxygenase cinnamate 4-hydroxylase (C4H). 41p-OH-Coumaric acid is the skeleton for lignan natural products.Analogously, deamination of tyrosine is facilitated by tyrosine ammonia-lyase (TAL). 41,42Cinnamic acid and p-OH-coumaric acid not only act as building blocks in lignans, but also serve as precursors to the structural polymer lignin, the second most abundant biopolymer on earth after cellulose. 43,44PAL/TAL utilizes 5-methylidene-imidazolone (MIO) as the cofactor that is formed from three adjacent glycine, serine and alanine.Briefly, attack of the substrate's amino group onto the MIO cofactor generates a reactive intermediate, in which deprotonation followed by cleavage of the amine produces the C=C bond. 42rom p-OH-coumaric acid, a common strategy is employed to generate CA, as well as the other two monolignols.This strategy involves an ATP-dependent 4-coumarate-CoA-ligase (4CL) to activate the C1carboxylate of the p-OH-coumaric acid via CoA ligation. 45,46With p-OH-coumaric acid also acting as an entry point for flavonoid biosynthesis, 4CL is indicated to channel the flux of p-OH-coumaric acid towards the respective pathways.In Arabidopsis thaliana, 4 isoforms of 4CL were identified, 2 of which were implicated in phenylpropanoid biosynthesis, 1 in flavonoid biosynthesis and 1 in piperine biosynthesis. 50-54p-OH-Coumaroyl CoA undergoes shikimate/quinate group transfer by hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyl transferase (HCT).HCT catalyzes the shikimation/quination of p-OH-coumaroyl CoA, which is hydroxylated by a cytochrome P450 monooxygenase C3'H and subsequently returned to the CoA ester by HCT. 49CT thus catalyzes a two-step reaction, yielding caffeoyl-CoA. 50Additionally, feruloyl CoA production may also proceed via 4CL-mediated CoA ligation of ferulic acid.Furthermore, O-methylation catalyzed by SAM dependent caffeate 3-O-methyltransferase (COMT) or caffeoyl-CoA-O-methyltransferase (CCoAOMT) leads to feruloyl CoA formation (Figure 3). 51,54 th the production of p-OH-coumaroyl CoA, caffeoyl CoA and feruloyl CoA being clarified, subsequent steps toward CA follow a common pattern.The respective CoA-ester is reduced by NADPH-dependent cinnamoyl-CoA reductase (CCR). 52,53Caffeoyl-CoA and feruloyl-CoA are readily reduced by CCR to generate the corresponding aldehyde intermediates: caffeyl aldehyde and coniferyl aldehyde, respectively.Caffeyl aldehyde may be converted to coniferyl aldehyde by COMT or further reduced to caffeyl alcohol by cinnamyl alcohol dehydrogenase (CAD) and subsequently O-methylated by COMT to generate CA.Reduction of coniferaldehyde by CAD naturally also results in the production of CA. 54 Additionally, reduction of p-OH-coumaroyl CoA yields p-OH-coumaraldehyde.C3-hydroxylation of p-coumaraldehyde has been shown in vitro, but there currently is no evidence supporting the results in planta.Therefore, p-OH-coumaraldehyde production is likely a shunt pathway. 50

Scaffold Generation Overview
Dimerization of monolignols proceeds in a phenolic oxidative coupling reaction facilitated by an oxidase, e.g.laccase, and dirigent protein (DIR), in which the oxidase generates a phenoxy radical and DIR induces stereoand regio-selective coupling of two monolignol radical species.Coupling of two coniferyl alcohol radicals gives rise to the primary lignan precursor, pinoresinol, other products including norlignans, neolignans are derived from coupling of other resonance forms of the phenoxy radical intermediates.The coupling mode leading to pinoresinol is depicted in Figure 4. Pinoresinol (the furofuran type) may then undergo consecutive reduction by pinoresinol lariciresinol reductase (PLR) to lariciresinol (furan-type) and secoisolariciresinol (dibenzylbutanetype). Secoisolariciresinol in turn is oxidized by secoisolariciresinol dehydrogenase (SDH) to form matairesinol.This pathway has been demonstrated in various lignan-producing plant species and can be described as the general lignan pathway.At each step, some intermediate flux is diverted from the general pathway and subjected to scaffold tailoring, which ultimately is the key to complexity generation in lignan biosynthesis.

Monolignol Dimerization
The dimerization process starts with the formation of the phenoxy radical intermediate.This process is enabled by oxidase enzymes, e.g, laccases and peroxidases. 55While the involvement of DIR has been studied and several isoforms have been characterized, the detailed mechanism remain to be elucidated. 568][59] In addition, the first plant laccase, ZmLac3 from Zea mays was characterized by Xie et al. in 2020. 60In the study, the structure of ZmLac3 was solved and found to exhibit three cupredoxin-like domains stabilized by disulfide bridges and harboring four Cu centers at the interface of the trimer.ZmLac3 was shown to exhibit substrate preference towards sinapyl alcohol, but it was able to use coniferyl alcohol and p-coumaryl alcohol.][63] Coupling of the monolignol phenoxy radical intermediates is facilitated by DIRs (Figure 5).It is in this step that stereo-and site-selectivity is imparted.DIR was first identified by in Forsythia suspensa.In the presence of (E)-coniferyl alcohol, exclusively (+)-pinoresinol-formation was observed, while racemic mixtures of pinoresinol and other coupling products were obtained when only laccase was present. 56,64The first crystal structure of DIR was characterized by Gasper et al. in 2016. 65AtDIR6 from Arabidopsis thaliana exhibited eight-stranded betabarrel topology forming a trimeric structure with well-defined and spatially separated substrate binding cavities, each possessing two pockets for the binding of the proposed radical intermediates.AtDIR6 was shown to exhibit enantio-complementary reactivity to FsDIR, yielding (-)-pinoresinol exclusively.In a recent study by Sattely et al. structural comparison of DIRs, PsDRR206 from Pisum sativum (for the production of (+)-pinoresinol) and AtDIR6 from Arabidopsis thaliana (for the production of (-)-pinoresinol), demonstrated previously postulated differential binding modes resulting in respective si-si or re-re coupling. 58In the same study PhDIR from Podophyllum hexandrum in conjunction with a fungal laccase TvLac from Trametes versicolor converted (E)coniferyl alcohol and synthetic CA-analogs with varied meta-substitution to (+)-pinoresinol and analogues.The regioselective coupling mediated by DIR not only facilitates the formation of the lignan-defining C8-C8' bond but is also the source of chirality in lignan biosynthesis.Overall, three new bonds (including one C-C and two C-O bonds) and four new stereocenters are generated in this single-step coupling -a feat unachievable by synthetic methods.While homotypic dimerization of coniferyl alcohol serves as the biosynthetic origin of the vast majority of lignan natural products, the scope of DIR-mediated coupling also includes other monolignols and likely extends to downstream intermediates of phenylpropanoid metabolism such as isoeugenol. 66,67egardless of substrate or coupling mode, the presence of para-hydroxyl moiety is a requirement of oxidase/DIR-enabled dimerization.It has been proposed that the hydroxyl group stabilizes the radical intermediate, therefore facilitating dimerization process. 56,57The involvement of oxidase enzymes in radical generation and delivery, as well as the mechanism of radical capture by DIR remains poorly understood.© AUTHOR(S) Figure 5.The proposed dimerization mechanism.

Reduction of Pinoresinol/Lariciresinol
Sequential reduction of the furan rings of pinoresinol to lariciresinol, then secoisolariciresinol is mediated by NADPH-dependent pinoresinol-lariciresinol-reductase (PLR) (Figure 6).Early investigations of PLR demonstrated the involvement of two isoforms of PLR, each carrying out one reduction in Forsythia intermedia. 68nvestigations of PLRs in lignan producing plant species, revealed varying degrees of substrate selectivity, with some PLRs can facilitate the reduction of both pinoresinol and lariciresinol.Therefore, the observed variations in substrate specificity of PLR likely play a role in controlling and diverting lignan flux in producing organisms. 69he phenolic para-OH group is important for PLR activity, thus revealing a possible strategy for the reaction mechanism.The reaction is proposed to proceed via a quinone methide intermediate, ring opening, followed by a hydride provided from NADPH to restore aromaticity. 70Biochemical characterization of PLR homologs using exogenously supplied racemic pinoresinol and lariciresinol has revealed that the PLR-catalyzed reduction is highly enantioselective.For example, PLR-Tp1 from Thuja plicata catalyzes stereoselective reduction of (+)pinoresinol and (+)-lariciresinol, while PLR-Tp2 reduces (-)-pinoresinol and (-)-lariciresinol. 70Though the observed enantiospecificity of PLR-Tp1 and PLR-Tp2 has been investigated using X-ray crystallographic analyses and site-directed mutagenesis approaches, the factors governing selectivity remain unclear. 71dditionally, the substrate specificity was investigated by Wu et al. via structural comparison of two PLR isoforms from Camellia sinensis. 72A variable loop region consisting of 13 amino acid residues was found to be crucial to substrate recognition.Recently, Xiao et al. characterized the structures of PLR isoforms, IiPLR1 (Isatis indigotica), AtPrR1 (Arabidopsis thaliana) and AtPrR2 (Arabidopsis thaliana), each with substrates pinoresinol and lariciresinol bound.In conjunction with biochemical assays, both substrate-and enantio-specificity were investigated.While IiPLR1 exhibited comparable efficiency toward both pinoresinol and lariciresinol, AtPrR1 showed significant preference for pinoresinol and AtPrR2 exclusively converted pinoresinol. 73By introducing mutations in the variable loop region, the substrate preference of IiPLR1 and AtPrRs could be switched.Though potential residues contributing to substrate enantiospecificity were proposed, their identity and the underlying mechanism could not be verified. 72,73

Dehydrogenation of Secoisolariciresinol
The next step in the lignan biosynthetic pathway involves the conversion of dibenzylbutane-type secoisolariciresinol to dibenzyllactone-type matairesinol.Selective sequential oxidation of one of the alcohols is facilitated by NAD(P)H dependent secoisolariciresinol-dehydrogenase (SDH) (Figure 7).The resulting carboxylic acid is then subjected to intramolecular cyclization resulting in lactone formation. 29Structural analysis of SDH isolated from Podophyllum peltatum revealed a homotetrameric structure with a highly conserved catalytic triad consisting of Ser153, Tyr167 and Lys171. 74,75In vitro biochemical assays with SDH from Forsythia intermedia and Podophyllum peltatum indicated higher activity in the presence of NADH instead of NADPH.Furthermore, incubation with racemic secoisolariciresinol resulted in selective formation of (-)-matairesinol. 75Overall, the general lignan biosynthetic pathway utilizes sequential enzymatic transformations to generate four different lignan scaffolds: furofuran, furan, dibenzylbutane and dibenzyllactone, in which a vast majority of lignan natural products originate from either one of the four intermediates.Notably, varying degrees of enantioand substrate-specificity divert the incoming flux of pinoresinol toward the diverse composition of lignan natural product observed in respective producer organisms.

Formation of Podophyllotoxin Core
Due to the pharmacological importance of podophyllotoxin, its biosynthesis has been the subject of extensive research and responsible genes have been largely identified.Herein, the biosynthetic pathway from matairesinol to deoxypodophyllotoxin will be detailed (Figure 8).The four enzymes responsible for the conversion of (-)-pluviatolide to (-)-deoxypodophyllotoxin in Sinopodophyllum hexandrum were first identified in 2015 by Lau and Sattely. 32The first transformation involves the conversion of (-)-matairesinol to (-)pluviatolide.Methylenedioxy (MDO) bridge formation in plants is associated with the cytochrome P450 monooxygenase family (CYP).CYPs compose a large and diverse enzyme superfamily, with members playing essential roles in plant secondary metabolism.For example, CYP719A1 from Coptis japonica catalzyed MDO formation in isoquinoline alkaloid biosynthesis. 76,77Leveraging massively parallel sequencing of Sinopodophyllum hexandrum and Podophyllum peltatum transcriptomes, Marquez et al. identified two P450s involved in podophyllotoxin biosynthesis, ShCYP719A23 and PpCYP719A24. 78In addition, other enzymes, e.g., non-heme iron oxygenases, have also been implicated in MDO bridge formation. 79In the next step, (-)pluviatolide is converted to (-)-bursehernin via O-methylation at C4'.It is followed by C5'-hydroxylation to yield (-)-5'-desmethyl-yatein and methylation to yield (-)-yatein. 80 The stereo-and regioselective C(sp 2 )-C(sp 3 ) bond formation between the benzylic C7' and aryl C6 yields (-)-deoxypodophyllotoxin and completes the tetracyclic core of the aryltetralin class.Iron and 2-oxoglutarate (Fe/2OG) dependent Sh2-ODD/ShDPS from Sinopodophyllum hexandrum facilitates stereo-and regioselective C(sp 2 )-C(sp 3 ) bond formation. 32.The mechanism of this transformation was elucidated in 2022 by Tang et al. using substrate-bound protein structures, biochemical assays with substrate analogs and chemical model studies. 81Briefly, the reaction is proposed to proceed via elimination of a hydrogen atom to generate a substrate radical.Following a carbocation at C7', C-C bond formation is likely induced by rotation of the benzodioxole moiety, positioning its C6 in close proximity to the C7'-centered carbocation (Figure 9).Furthermore, DPS was shown to convert both (+)-and (-)-yatein.Interestingly, the stereochemical conformation of the substrate does not impact the stereo-specificity of C-C bond formation. 81

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© AUTHOR(S) Figure 10.Tailoring enzymes in the formation of podophyllotoxin and derivatives.

Scaffold Tailoring in Sesamin Biosynthesis
A majority of furofuran lignans are derived from pinoresinol, as well as pinoresinol analogs stemming from related monolignol dimerization. 22,39,83Among furofuran lignans, sesamin is one of the most well-known and commercially relevant.It is mainly produced by Sesamum sp., e.g., Sesamum indicum. 23,84Sesamin is produced from pinoresinol through formation of two methylenedioxy bridges (Figure 11).The pathway from pinoresinol to sesamin was first proposed in 1998 by Lewis et al. 84 In the study, a cytochrome P450 monooxygenase was shown to convert pinoresinol to piperitol.Further conversion to sesamin was not observed, hence the enzyme was termed piperitol synthase (PS) and the presence of a second enzyme, sesamin synthase (SS) was postulated. 84Ono et al. later reported the identification and characterization of three SiP450 proteins, encoded by genes CYP81Q1 from Sesamum indicum, CYP81Q2 from Sesamum radiatum and CYP81Q3 from Sesamum alatum.Both recombinant proteins of CYP81Q1 and CYP81Q2 were shown to possess dual (+)-piperitol/(+)sesamin forming activity, thus implicating them as (+)-piperitol/(+)-sesamin synthases (PSS). 85Likely, (+)piperitol is released from the enzyme active site after the first catalysis and is subsequently recaptured for the second catalysis (Figure 11).However, the mechanism of dual catalysis remains to be fully elucidated.On the other hand, CYP81Q3 showed no activity, coinciding with the absence of (+)-sesamin in S. alatum.

Glycosylation
Glycosylation represents one of the most common group transfer reactions in natural product biosynthesis to diversify natural product structures and introduction of a sugar moiety improves solubility and reactivity of the corresponding aglycone. 29,86,87In plants, glycosylation is typically facilitated by uridine diphosphate (UDP)dependent glycosyltransferases (UGTs).In lignan biosynthesis, glycosylation is also commonly used.AtUGT71C1 from Arabidopsis thaliana was shown to perform monoglycosylation on both (+)-lariciresinol and (+)-pinoresinol with comparable efficiency.On the other hand, IiUGT71B5 from Isatis indigotica facilitates mono-and diglycosylation on pinoresinol only (Figure 12).Additionally, LuUGT74S1 from Linum usitatissimum could utilize (-)-secoisolariciresinol as a substrate to catalyze mono-and di-glycosylation. 88Another prominent example of a lignan-glucoside is found in etoposide.The disaccharide moiety in etoposide is important to its topoisomerase II inhibition activity. 89,90Glycosylation of the etoposide aglycone, (-)-4'-desmethylepipodophyllotoxin, has been achieved by leveraging the substrate promiscuity of non-native UGTs (Figure 10). 91

Arylnaphthalene Lignans
Arylnaphthalene lignans are produced by plant species of various genera, including Haplophyllum, Justicia, Larrea and Phyllantus.3][94] Justicidins and diphyllin are prominent examples of the subtype and have been © AUTHOR(S) investigated as putative remedies for topical inflammatory diseases such as psoriasis.Structurally, the arylnaphthalene scaffold is defined by a biaryl bond connecting naphthalene and phenyl moieties.Consequently, arylnaphthalene lignans lack the chiral centers characteristic of other lignan subclasses.Instead, the rotatory hindrance of the sp 2 -sp 2 biaryl bond induces axial chirality and consequently the ability of forming atropisomers.Atropisomerism is rarely observed in nature, thus they have garnered considerable research interest in recent years. 95,96To date, the biosynthetic formation of the arylnaphthalene-type lignan scaffold remains a mystery.From a biosynthetic viewpoint, it appears likely that arylnaphthalene lignans are derived from sequential dehydration of corresponding aryltetralin precursor.

Dibenzylcyclooctadiene Lignans
A large number of dibenzylcyclooctadiene-type have been isolated and characterized.The structural features and promising bioactivities of representative compounds have elicited significant research interest. 17,20,98,99However, like arylnaphthalene lignans, the genes associated with the biosynthetic transformations leading to their formation remain unknown.However, several possible pathway intermediates have been proposed to provide some insight into early steps of Dibenzylcyclooctadiene Lignans (DBO) biosynthesis. 39,100nterestingly, all known and characterized DBO-lignans have been isolated from the Schisandraceae family. 18,19,68,101The lack of genomic sequencing data of Schizandera sp. has made the identification of enzymes involved in their biosynthesis elusive.Recent advances in verrucosin biosynthesis in Schizandera sp., have implicated it to be an on-pathway intermediate toward DBO-type lignans.In contrast to using coniferyl alcohol as the precursor, verrucosin is likely resulting from dimerization of (E)-isoeugenol.Isotope labeling studies by Lopes et al. observed verrucosin formation in Virola surinamensis using labeled (E)-isoeugenol. 66Furthermore, Dexter et al. identified an acyltransferase (PhCFAT) in petunias responsible for the acetylation of coniferyl alcohol to yield coniferyl acetate. 67A novel NADPH-dependent isoeugenol synthase (PhIGS1) was shown to convert only coniferyl acetate, but not coniferyl alcohol, to (E)-isoeugenol.More recently, Dong et al. identified DIR and ScCFAT genes in S. chinensis. 100,102Taken together, a possible pathway for verrucosin formation was proposed (Figure 14).(E)-Isoeugenol is speculated to undergo dimerization to verrucosin facilitated by an auxiliary oxidase and ShDIR, which in turn could be converted to dihydroguiaretic acid via a PLR-like reductase.Subsequently, cytochrome P450s and O-methyltransferases may then form pregomisin.

Figure 1 .
Figure 1.The structural features, nomenclature and classification of lignans.

Figure 4 .
Figure 4. Formation of lignans: scaffold generation through coupling and other tailoring reactions.

Figure 9 .
Figure 9. Proposed mechanism of an Fe/2OG enzyme catalyzed C-C bond formation.