title: Demonstration that limonene is the first cyclic intermediate in the biosynthesis of oxygenated p-menthane monoterpenes in Mentha piperita and other Mentha species.
abstract: 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. The time course of incorporation of [U-14C]sucrose into the monoterpenes of M. piperita shoot tips was consistent with the initial formation of limonene and its subsequent conversion to menthone via pulegone. d,l-[9-3H]Limonene and [9,10-3H]terpinolene were prepared and tested directly as precursors of oxygenated p-menthane monoterpenes in M. piperita shoot tips. Limonene was readily incorporated into pulegone, menthone, and other oxygenated derivatives, whereas terpinolene was not appreciably incorporated into these compounds. Similarly, d,l-[9-3H]limonene was specifically incorporated into pulegone in Mentha pulegium and into the C-2-oxygenated derivative carvone in Mentha spicata, confirming the role of this olefin as the essential precursor of oxygenated p-menthane monoterpenes. Soluble enzyme preparations from the epidermis of immature M. piperita leaves converted the acyclic terpenoid precursor [1-3H]geranyl pyrophosphate to limonene as the major cyclic product, providing a further indication that this olefin plays a central role in the formation of oxygenated monoterpenes in Mentha. No free intermediates were detected in the cyclization of geranyl pyrophosphate to limonene, suggesting that the olefin is the first cyclic intermediate to arise in the pathway, and resolution of the biosynthetic limonene, by crystallization of the derived d- and l-carvoximes, indicated an enantiomer mixture nearly identical to that isolated from the leaf oil.
mesh_terms: D002474:Cell-Free System; D003500:Cyclization; D053138:Cyclohexenes; D000077222:Limonene; D008610:Menthol / Q000031:analogs & derivatives*; D009821:Oils / Q000032:analysis; D010084:Oxidation-Reduction; D010946:Plants, Medicinal / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D013729:Terpenes / Q000096:biosynthesis*
chemical_list: D053138:Cyclohexenes; D009821:Oils; D011106:Polyisoprenyl Phosphates; D013729:Terpenes; D008610:Menthol; D013395:Sucrose; C015234:geranyl pyrophosphate; D000077222:Limonene