Stereochemistry at C-1 of geranyl pyrophosphate and neryl pyrophosphate in the cyclization to (+)- and (-)-bornyl pyrophosphate.

(1R)-1-3H-labeled and (1S)-1-3H-labeled geranyl pyrophosphate and neryl pyrophosphate were prepared from the corresponding 1-3H-labeled aldehydes by a combination of enzymatic and synthetic procedures. Following admixture with the corresponding 2-14C-labeled internal standard, each substrate was converted to (+)-bornyl pyrophosphate and (-)-bornyl pyrophosphate by cell-free enzyme preparations from sage (Salvia officinalis) and tansy (Tanacetum vulgare), respectively. Each pyrophosphate ester was hydrolyzed, and the resulting borneol was oxidized to camphor.

Metabolism of Monoterpenes : Evidence for the Function of Monoterpene Catabolism in Peppermint (Mentha piperita) Rhizomes.

l-Menthone of peppermint leaves is reduced to d-neomenthol which is glucosylated and transported to the rhizome, whereupon the beta-d-glucoside is hydrolyzed, the aglycone oxidized back to l-menthone, and this ketone converted to l-3,4-menthone lactone. l-[G-(3)H]-3,4-Menthone lactone and its labeled progenitors, when incubated with excised mint rhizomes, gave rise to nonvolatile lipids as well as polar metabolites. The lipids thus generated consisted of labeled squalene and phytosterols in the nonsaponifiable fraction and C(14)-C(26) fatty acids in the saponifiable fraction.

Metabolism of monoterpenes: oxidation of isopiperitenol to isopiperitenone, and subsequent isomerization to piperitenone by soluble enzyme preparations from peppermint (Mentha piperita) leaves.

Soluble enzyme extracts from peppermint leaves, when treated with polystyrene resin to remove endogenous monoterpenes and assayed with unlabeled substrates coupled with capillary gas-liquid chromatographic/mass spectrometric detection methods, were shown to oxidize isopiperitenol to isopiperitenone, and to isomerize isopiperitenone to piperitenone. The enzymes responsible for the monoterpenol dehydrogenation and the subsequent allylic isomerization were separated and partially purified by chromatography on Sephadex G-150, and were shown to have molecular weights of approximately 66,000 and 54,000, respectively. The general properties of the NAD-dependent dehydrogenase were examined, and specificity studies indicated that a double bond adjacent to the carbinol carbon was a required structural feature of the monoterpenol substrate.

Biosynthesis of monoterpenes: demonstration of a geranyl pyrophosphate:(-)-bornyl pyrophosphate cyclase in soluble enzyme preparations from tansy (Tanacetum vulgare).

Tansy (Tanacetum vulgare L.) produces an essential oil containing the optically pure monoterpene ketone, (-)-camphor, as a major constituent. A soluble enzyme preparation from immature leaves of this plant converts the acyclic precursor [1-3H]geranyl pyrophosphate to the bicyclic monoterpene alcohol borneol in the presence of MgCl2, and oxidizes a portion of the borneol to camphor in the presence of a pyridine nucleotide.

Biosynthesis of ascaridole: iodide peroxidase-catalyzed synthesis of a monoterpene endoperoxide in soluble extracts of Chenopodium ambrosioides fruit.

Ascaridole, an asymmetric monoterpene endoperoxide with anthelmintic properties, occurs as a major constituent (60-80%) in the volatile oil of American wormseed fruit (Chenopodium ambrosioides: Chenopodiaceae), and as a lesser component in the leaf pocket oil of the boldo tree (Peumus boldus: Monimiaceae). Determination of optical activity and chromatographic resolution of naturally occurring ascaridole, and several synthetic derivatives, showed that both wormseed and boldo produce ascaridole in racemic form. The biosynthesis of ascaridole from the conjugated, symmetrical diene alpha-terpinene (a major component of the oil from wormseed) was shown to be catalyzed by a soluble iodide peroxidase isolated from homogenates of C.

Metabolism of Monoterpenes : Early Steps in the Metabolism of d-Neomenthyl-beta-d-Glucoside in Peppermint (Mentha piperita) Rhizomes.

Previous studies have shown that the monoterpene ketone l-[G-(3)H] menthone is reduced to the epimeric alcohols l-menthol and d-neomenthol in leaves of flowering peppermint (Mentha piperita L.), and that a portion of the menthol is converted to menthyl acetate while the bulk of the neomenthol is transformed to neomenthyl-beta-d-glucoside which is then transported to the rhizome (Croteau, Martinkus 1979 Plant Physiol 64: 169-175). Analysis of the disposition of l-[G-(3)H]menthone applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis and transport of the monoterpenyl glucoside to be determined, and gave strong indication that the glucoside was subsequently metabolized in the rhizome.

Development and Essential Oil Content of Secretory Glands of Sage (Salvia officinalis).

Scanning electron microscopy of sage (Salvia officinalis L.) leaves confirmed the presence of two basic types of glandular trichomes consisting of a capitate stalked form containing a multicellular stalk and surmounted by a unicellular secretory head, and a capitate sessile form containing a unicellular stalk and unicellular, or multicellular, secretory head. In the latter type, secretory activity and filling of the subcuticular cavity may begin at virtually any stage of the division cycle affording fully developed glands containing from one to twelve cells in the secretory head.

Cyclization of farnesyl pyrophosphate to the sesquiterpene olefins humulene and caryophyllene by an enzyme system from sage (Salvia officinalis).

A soluble enzyme preparation obtained from sage (Salvia officinalis) leaves was shown to catalyze the divalent metal-ion dependent cyclization of trans, trans-farnesyl pyrophosphate to the macrocyclic sesquiterpene olefins humulene and caryophyllene. The identities of the biosynthetic products were confirmed by radiochromatographic analysis and by preparation of crystalline derivatives, and the specificity of labeling in the cyclization reaction was established by chemical degradation of the olefins derived enzymatically from [1-3H2]farnesyl pyrophosphate. These results constitute the first report on the cyclization of farnesyl pyrophosphate to humulene and caryophyllene, two of the most common sesquiterpenes in nature, and the first description of a soluble sesquiterpene cyclase to be isolated from leaves of a higher plant.

Purification of monoterpenyl glycosides by gel-permeation and hydrophobic-interaction chromatography on polyacrylamide (Bio-Gel P-2).

Hydrophobic interaction of the aglycone of monoterpenyl glycosides with the polyacrylamide matrix of Bio-Gel P-2 greatly retards the elution of these substances when chromatographed in dilute aqueous sodium chloride. This hydrophobic interaction is eliminated by inclusion of 15% acetonitrile in the eluant, thereby permitting conventional gel-permeation chromatography. Combination of these techniques by sequential chromatography on the same Bio-Gel column, in the hydrophobic interaction mode followed by the gel-permeation mode, provides a simple, yet mild and highly selective procedure for the purification of monoterpenyl glycosides from crude plant extracts.

Metabolism of monoterpenes: lactonization of (+)-camphor and conversion of the corresponding hydroxy acid to the glucoside-glucose ester in sage (Salvia officinalis).

The bicyclic monoterpene ketone (+)-camphor is a major constituent (up to 26%) of the volatile oil of immature sage (Salvia officinalis L.) leaves; however, as the plant matures the content of this ketone declines in the fully expanded leaves (to about 65% of maximum) as does the overall yield of oil (to roughly 60% of maximum). Examination of the metabolism of (+)-[G-3H]camphor in discs prepared from mature leaves of flowering sage plants revealed that this ketone was converted to a water-soluble metabolite which on chromatographic analysis proved to be considerably more polar than a simple monoterpenyl glycoside.

Pinene cyclases I and II. Two enzymes from sage (Salvia officinalis) which catalyze stereospecific cyclizations of geranyl pyrophosphate to monoterpene olefins of opposite configuration.

A soluble enzyme preparation from immature sage (Salvia officinalis) leaves has been shown to catalyze the cation-dependent cyclization of geranyl pyrophosphate to the isomeric monoterpene olefins (+/-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of camphene and limonene (Gambliel, H., and Croteau, R. (1982) J.

Demonstration that limonene is the first cyclic intermediate in the biosynthesis of oxygenated p-menthane monoterpenes in Mentha piperita and other Mentha species.

The volatile oil of mature Mentha piperita (peppermint) leaves contains as major components the oxygenated p-menthane monoterpenes l-menthol (47%) and l-menthone (24%) as well as very low levels of the monoterpene olefins limonene (1%) and terpinolene (0.1%), which are considered to be probable precursors of the oxygenated derivatives. Immature leaves, which are actively synthesizing monoterpenes, produce an oil with comparatively higher levels of limonene (approximately 3%), and isolation of the pure olefin showed this compound to consist of approximately 80% of the l-(4S)-enantiomer and approximately 20% of the d-(4R)-enantiomer.

Metabolism of Monoterpenes: Conversion of l-Menthone to l-Menthol and d-Neomenthol by Stereospecific Dehydrogenases from Peppermint (Mentha piperita) Leaves.

The monoterpene ketone l-menthone is specifically converted to l-menthol and l-menthyl acetate and to d-neomenthol and d-neomenthyl-beta-d-glucoside in mature peppermint (Mentha piperita L. cv. Black Mitcham) leaves.

Demonstration of the Intercellular Compartmentation of l-Menthone Metabolism in Peppermint (Mentha piperita) Leaves.

The metabolism of l-menthone, which is synthesized in the epidermal oil glands of peppermint (Mentha piperita L. cv. Black Mitcham) leaves, is compartmented; on leaf maturity, this ketone is converted to l-menthol and l-menthyl acetate in one compartment, and to d-neomenthol and d-neomenthyl glucoside in a separate compartment.

Metabolism of Monoterpenes : EVIDENCE FOR COMPARTMENTATION OF l-MENTHONE METABOLISM IN PEPPERMINT (MENTHA PIPERITA) LEAVES.

Previous studies have shown that the monoterpene ketone l-[G-(3)H]-menthone is reduced to the epimeric alcohols l-menthol and d-neomenthol in leaf discs of flowering peppermint (Mentha piperita L.), and that a portion of the menthol is converted to menthyl acetate while the bulk of the neomenthol is transformed to neomenthyl-beta-d-glucoside (Croteau, Martinkus 1979 Plant Physiol 64: 169-175). The metabolic disposition of the epimeric reduction products of the ketone, which is a major constituent of peppermint oil, is highly specific, in that little neomenthyl acetate and little menthyl glucoside are formed.

Relationship of Camphor Biosynthesis to Leaf Development in Sage (Salvia officinalis).

The camphor content of sage (Salvia officinalis L.) leaves increases as the leaves expand, and the increase is roughly proportional to the number of filled peltate oil glands which appear on the leaf surface during the expansion process. (14)CO(2) is more rapidly incorporated into camphor and its direct progenitors in expanding leaves than in mature leaves, and direct in vitro measurement of the key enzymes involved in the conversion of geranyl pyrophosphate to camphor indicates that these enzymes, including the probable rate-limiting cyclization step, are at the highest levels during the period of maximum leaf expansion.

Metabolism of Monoterpenes: Demonstration of (+)-Neomenthyl-beta-d-Glucoside as a Major Metabolite of (-)-Menthone in Peppermint (Mentha Piperita).

(-)-Menthone, the major monoterpene component of the essential oil of maturing peppermint (Mentha piperita L.) leaves (6 micromoles per leaf) is rapidly metabolized at the onset of flowering with a concomitant rise in the level of (-)-menthol (to about 2 micromoles per leaf). Exogenous (-)-[G-(3)H]menthone is converted into (-)-[(3)H]menthol as the major steam-volatile product in leaf discs in flowering peppermint (10% of incorporated tracer); however, the major portion of the incorporated tracer (86%) resided in the nonvolatile metabolites of (-)-[G-(3)H]menthone.