Lignan Biosynthesis

Lignans are synthesized from the oxidative coupling of monolignol radicals (see Section 10 in this chapter, and Chapter 1, Section 3.11). The monolignol radicals are generated through the action of laccases (E.C. 1.10.3.2) or peroxidases (E.C. 1.11.1.7; see also Chapter 2, section 1.8). Unlike lignin, however, lignans are optically active, and typically only one enantiomer is present in a given species. This means that the coupling between the monolignols is under regio-chemical control, whereby both the coupling site and the orientation of the two monomers are controlled. Analysis of Forsythia intermedia stem extracts, which accumulate high levels of the lignan (+)-pinoresinol (3.82) resulted in the purification of a protein that has no catalytic activity, but that is able to stipulate the formation of (+)-pinoresinol from two coniferyl alcohol (3.79) radicals (Davin et al., 1997). This protein was referred to as 'dirigent protein'. It is thought to hold the two coniferyl alcohol radicals in a specific configuration during bond formation so that both the position of the bond (8-8) and the conformation are controlled (Figure 3-10). Once the bond is formed, intramolecular cyclization results in (+)-pinoresinol (3.82). This particular dirigent protein was only able to recognize coniferyl alcohol radicals and none of the other monolignol radicals. Since the initial discovery of this protein from Forsythia intermedia, homology searches in sequence databases have revealed the existence of additional genes encoding putative dirigent proteins, from a variety of species (Gang et al., 1999; Davin and Lewis, 2000). Recently, evidence for the existence of dirigent proteins able to direct the formation of 8-0-4' linkages between coniferyl alcohol and sinapyl alcohol was reported (Lourith et al., 2005).

Figure 3-10. Dirigent-protein mediated formation of (+)-pinoresinol from two coniferyl alcohol radicals. The formation of the ring results from intramolecular cyclization.

Figure 3-10. Dirigent-protein mediated formation of (+)-pinoresinol from two coniferyl alcohol radicals. The formation of the ring results from intramolecular cyclization.

Lignans represent an extremely diverse group of compounds. This is the result of both structural diversity and stereo-selective biosynthesis. One particular plant species generally makes only one enantiomer of a particular compound. The other enantiomer may be synthesized by a different species. As a consequence, it is virtually impossible to summarize the biosynthesis of lignans in general. Instead, the focus here will be on the biosynthesis of the lignan podophyllotoxin in a number of different plant species, as an illustration of the different biosynthetic routes that can be used to synthesize the same compound.

Podophyllotoxin (3.86) is used as an ectopic antiviral agent in the treatment of venereal warts. It is too cytotoxic to be ingested, but after chemical modifications podophyllotoxin can be used as a powerful anti-cancer drug in the treatment of small cell lung cancer, acute leukemia, and non-Hodgkin's lymphoma (Canel et al., 2000). Podophyllotoxin is isolated from the plant Podophyllum peltatum, commonly known as Mayapple (also referred to as Devils's apple or hog apple), or from the related species Podophyllum hexandrum (Himalayan mayapple). In order to be able to produce podophyllotoxin without depleting the natural populations of these wild species, attempts have been made to produce podophyllotoxin in tissue culture. In this context it is important to know the biosynthetic route to podophyllotoxin production, so that lines that produce high levels of the relevant biosynthetic enzymes can be selected. Broomhead et al. (1991) investigated the biosynthesis of podophyllotoxin in Podophyllum hexandrum using radioactive feeding experiments in whole plants. They proposed the biosynthetic route displayed in Figure 3-11. This shows how two coniferyl alcohol (3.79) radicals are coupled in a stereo-selective manner to form matairesinol (3.83). Subsequent hydroxylation and methylation of C5' and lactone ring formation at C3 and C4 gives yatein

(3.84). Ring closure results in the formation of deoxypodophyllotoxin

(3.85), which is hydroxylated to yield podophyllotoxin (3.86).

In addition to podophyllotoxin itself, closely related compounds, such as 5-methoxypodophyllotoxin (3.89), can be used as a precursor for anti-cancer drugs. Linum species (flax, linseed) can produce podophyllotoxin or substituted podophyllotoxins in tissue culture with yields of up to 0.35% of dry weight. The biosynthesis of 5-methoxypodophyllotoxin in Linum flavum was investigated by Xia et al. (2000), whereas Seidel et al. (2002) investigated podophyllotoxin (3.86) production in cell cultures of Linum album.

Xia et al (2000) proposed a biosynthetic route starting coniferyl alcohol (3.79) and subsequent formation of (+)-pinoresinol (3.82), as shown in Figure 3-12. The enzyme pinoresinol/lariciresionol reductase converts this compound to (+)-lariciresinol (3.87), and then to (-)-secoisolariciresionol (3.88). The enzyme secoisolariciresinol dehydrogenase converts (3.88) into (-)-matairesinol (3.83). The conversion from (-)-matairesinol (3.83) to podophyllotoxin (3.86) is likely to be similar to the route shown in Figure 3-11.

110 h3co.

oh oh

oh oh h3co o

o h3co h3co och3

och3

ho h3co ho och3

h3co och3

h3co och3

o ho ho o h3co h3co och3

och3

och3

och3

Figure 3-11. Biosynthesis of podophyllotoxinin in Podophyllum hexandrum, according to Broomhead et al. (1991). The dotted arrow refers to a series of reactions that have not yet been fully elucidated: formation of a methylene dioxybridge, and hydroxylation and methylation reactions.

OCH,

OCH,

OCH,

OCH,

OCH,

OCH,

NADPH NADP+

H,CO

H,CO

NAD+ NADH

OCH,

NAD+ NADH

OCH,

OCH,

H,CO

OCH,

H,CO HO

H,CO HO

OCH,

H,CO

H,CO

OCH,

OCH,

OCH,

OCH,

Figure 3-12. Biosynthesis of 5-methoxypodophyllotoxinin in Linum flavum, according to Xia et al. (2000). (a) pinoresinol/lariciresionol reductase, and (b) secoisolariciresinol dehydrogenase. The enzymes catalyzing steps c-g have not yet been elucidated, but involve (c) hydroxylation of C7, (d) ring closure, (e) hydroxylation of C5, (f) methylation of the OH-group on C5, and (g) formation of a methylene dioxy bridge. The exact order of these reactions is also still unknown.

Synthesis of podophyllotoxin (3.86) in cell culture of Linum album results in yields comparable to those of the most efficient tissue cultures of Podophyllum hexandrum. In order to further improve L. album cultures, Seidel et al. (2002) investigated the biosynthesis of podophyllotoxin (3.86). They fed a number of labeled compounds that to L. album cell cultures to identify which of these compounds could be used as precursors to podophyllotoxin. They determined that the substitution pattern on the benzene ring is critical. The substitution has to be either 3-methoxy, 4-hydroxy, as in ferulic acid (3.33), or, alternatively, 3,4-methylenedioxycinnamic acid (3.90) can serve as precursor. The precursor of podophyllotoxin in L. album appears to be deoxypodophyllotoxin (3.83), based on the higher level of isotope incorporation in the latter compound. This means that 7-hydroxymatairesinol, the precursors of 5-methoxypodophyllotoxin in L. flavum (Xia et al., 2000), is not a precursor of podophyllotoxin in L. album.

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