Biosynthesis of C5 — C20Terpenoid Compounds

BY J. R. HANSON

1 Introduction

This chapter for 1972 follows the pattern oflast year's Report. During the year a number of reviews have appeared which discuss different aspects of terpenoid biosynthesis.

2 Mevalonic Acid

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase catalyses the two-step reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonic acid using NADPH. The hemithioacetal addition compound of mevaldic acid and coenzyme A is a possible intermediate. Both steps involve hydrogen transfer from the 4a (4R) position of NADPH. The mevaldic acid-coenzyme A hemithioacetal addition compound is a good substrate for HMG-CoA reductase. The hydrogen atom which is transferred in this step has been shown to appear at the 5-pro-S-position in the resulting mevalonic acid. This is in contrast to the stereochemistry of mevaldate reductase in which a 5-pro-R hydrogen atom is introduced. (see Scheme 1)

Cell-free extracts and acetone powder preparations from Agave americana have been shown to phosphorylate mevalonic acid to give phosphomevalonic acid and thence pyrophosphomevalonic acid at an optimum pH of 7.0. Glutathione and mercaptoethanol enhance the activity of these preparations. Tracer studies with tissue cultures derived from Tanacetum vulgare have revealed 8 the formation of phosphomevalonic acid, pyrophosphomevalonic acid, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, and the incorporation of mevalonic acid into monoterpenes. However, the mono-terpene components of the tissue culture differed from those of the whole plant, sabinene being formed rather than isothujone.

Artificial substrates have been studied in systems that form squalene and sterols. Incubation of trans-3-methyl[1,1-3H2]pent-2-enyl pyrophosphate and [1-14C]isopentenyl pyrophosphate with rat liver homogenates gave 1-methyl-squalene and 1,24-dimethylsqualene. However, the sterol fraction contained only 27-methyl-lanosterol and 27-methylcholesterol, indicating greater selectivity in either the epoxidation or cyclization stages of this biosynthesis. The substrate specificity of farnesyl pyrophosphate synthetase from pumpkin fruit has also been studied 10 with artificial allylie pyrophosphates. trans-3-Methyl-undec-2-enyl-, trans-3-methyldodec-2-enyl-, and trans-3-methyltetradec-2-enyl-pyrophosphates were assayed in the enzymatic reaction with isopentenyl pyrophosphate. The tetradecenyl pyrophosphate was inactive. Replacement of the methyl group by ethyl, as in trans-3-ethylhept-2-enyl-, trans-3-ethyloct-2-enyl-, and trans-3-ethyldec-2-enyl-pyrophosphates, gave reactive substrates. However, chain branching in the alkyl residues gave inactive substrates. 3-Ethylbut-3-enyl pyrophosphate also acts 11 as a substrate for farnesyl pyrophosphate synthetase with dimethylallyl pyrophosphate or geranyl pyrophosphate as the starter unit to afford homologues of farnesyl pyrophosphate.

3 Hemiterpenoids

Tryptophan, alanine, and mevalonic acid have been established in earlier work as biosynthetic precursors of echinulin (1). Cyclo-L-alanyl-L-trypto-phanyl (2) is a further intermediate whose isoprenylation has been studied. A cell-free system has been prepared from Aspergillus amstelodami which catalyses the transfer of one isoprene unit from dimethylallyl pyrophosphate to the cyclo-L-alanyl-L-tryptophanyl moiety. An enzyme system has been partially purified from cell-free extracts of E. coli which catalyses the synthesis of N6-(3,3-dimethylallyl)adenosine in transfer RNA.

Further studies have been reported on the biosynthesis of the hemiterpenoid furanocoumarins. A coumarin with a dimethylallyl group adjacent to a hydroxy-group figures in most of the biogenetic speculation concerning the origin of the furan ring in furanocoumarins. Demethyl[l'-14C]suberosin (3) is incorporated by the fruits of Angelica archangelica into the linear furanocoumarins such as bergapten (6), imperatorin (7), and isoimperatorin (8). Degradation of the bergapten established that incorporation occurred without randomization. Just as marmesin (4) acts as a precursor of psoralen (5), bergapten (6), and xanthotoxin (9), so it also acts as a precursor of rutaretin (10) in Ruta graveolens. 7-Hydroxyumbelliferone is the most effective general precursor of the coumarin portion of these furanocoumarins.

A thorough review has appeared on the biosynthesis of the ergot and associated alkaloids.

4 Monoterpenoids

A comprehensive and critical review, covering the literature to April 1971, on the biosynthesis and metabolism of the monoterpenes has appeared.

Mevalonic acid is often incorporated selectively into the second isoprene unit of the monoterpenes. This is attributed to compartmentalization effects in the biosynthesis of dimethylallyl pyrophosphate as a starter unit. 3,3-Dimethyl-acrylic acid has been suggested as an alternative precursor. However, a study of its incorporation into (+)-pulegone (11) by Mentha pulegium indicated extensive randomization of the label and hence decomposition and resynthesis. Further evidence for compartmentalization effects and the presence of an endogenous dimethylallyl pyrophosphate pool participating in monoterpene biosynthesis in vegetative tissue has come from the study of the incorporation of [14C]carbon dioxide and [14C]glucose into the monoterpenes of peppermint, Mentha piperita. The pulegone (11), derived from 14CO2 after different time intervals, was degraded and 90% of the radioactivity was found in the second isoprene unit (i.e. derived directly from IPP). Sucrose co-administered with [2-14C]mevalonic acid to peppermint cuttings enhances the incorporation of mevalonate into monoterpenes, indicative of energy requirements for this biosynthesis.

The isolation and properties of a monoterpene reductase from rose petals have been described. Geraniol and nerol were reduced by a solubilized enzyme preparation to give citronellol. The co-factor requirement was filled only by NADPH and the system had an optimum pH of 8. It was inhibited by p-chloromercuriophenylsulphonic acid, suggesting the presence of an S — H group near the active site.

Salvia officinalis has been shown to specifically incorporate [2-14C]geraniol into (-)-camphor (12) and (-)-borneol (13) (0.5 × 10-3 % and 3.3 × 10-3%) such that the tracer is incorporated into C-2 of both monoterpenes. This work contains a salutary discussion of the dangers of relying solely on g.l.c. purification of terpenes for...