1 2. Biosynthesis of Natural Products - Terpene Biosynthesis 2.1 Introduction Terpenes are a large and varied class of natural products, produced primarily by a wide variety of plants, insects, microoroganisms and animals. They are the major components of resin, and of turpentine produced from resin. The name "terpene" is derived from the word "turpentine". Terpenes are major biosynthetic building blocks within nearly every living creature. Steroids, for example, are derivatives of the triterpene squalene. When terpenes are modified, such as by oxidation or rearrangement of the carbon skeleton, the resulting compounds are generally referred to as terpenoids. Some authors will use the term terpene to include all terpenoids. Terpenoids are also known as Isoprenoids. Terpenes and terpenoids are the primary constituents of the essential oils of many types of plants and flowers. Essential oils are used widely as natural flavor additives for food, as fragrances in perfumery, and in traditional and alternative medicines such as aromatherapy. Synthetic variations and derivatives of natural terpenes and terpenoids also greatly expand the variety of aromas used in perfumery and flavors used in food additives. Recent estimates suggest that over 30'000 different terpenes have been characterized from natural sources. Early on it was recognized that the majority of terpenoid natural products contain a multiple of 5C-atoms. Hemiterpenes consist of a single isoprene unit, whereas the monoterpenes include e.g.: Monoterpenes CH2OH CHO CH2OH OH Myrcens Limonens Nerol Geraniol Citral Menthol O CHO O Camphor α-Pinene Citronellal Terpenes with 15 C-atoms are known as sesquiterpenes : Sesquiterpenes O CH2OH Farnesol Bisabolene Cadinene Selinene Vetivone OH HO COOH COOMe OH Abscisic acid O (Phytohormone) O Patchoulol (Perfume) O O Pentalenolactone (Antibiotic) The terpenes containing, or originating from precursors, containing 20 C-atoms are known as diterpenes, those with 30 C-atoms as triterpenes and those with 40 C-atoms as tetraterpenes : 2 Diterpenes Ph AcO O OH O NH O CH2OH Phytol Ph O O OH H CH2OH OAc Vitamin A OH OBz (Retinol) Taxol (anti-cancer) OH Giberellic acid (Phytohormone) O O Casbene HO (Phytoalexin) COOH Triterpene Squalene COOH O HO CH2O H O OH H H H H H H H H H HO HO OH O H Cholesterol Cholic acid Cortisone (Membrane component) (Hormone) Testosterone OH OH O (Hormone) Östradiol Progesterone (Hormone) (Hormone) H H H H H H H H H O HO O In contrast to other classes of terpenes that vary greatly in structure and molecular size, the steroids constitute a family of terpenes with a common structural feature, namely, the steroid ring system: Tetraterpene ß-Carotene (Pigment, Provitamin A) 3 Mixed origin Me O Plastoquinone Me (Electron transport) Chlorophyll-a N N (Photosynthesis) Mg N N O 18 Me Me OH O O COOMe O Tetrahydrocannabinol O C H (Cannabis sativa) 5 11 Polymer Rubber (Heva brasilensis) OH 500-5000 Ruzicka (ETH-ZH) recognized already in the 1920's that most terpenes appear to be constructed from a multiple of linked isoprene units. This is called the isoprene rule. The isoprene rule (cf. Birch, Polyketide Hypothesis) was of great value also in the structure determination of new terpenoids isolated from Nature. However, isoprene itself is not the building block used by Nature to construct terpenes. O OH Camphor OH CH2OH Grandisol Menthol Vitamin A Cadinene 2.2 The Mevalonate Pathway It was only much later (ca. 1955) shown that the biosynthesis of terpenes does indeed occur starting from isoprene-like C5 building blocks. Labelling experiments, using 14C-labelled acetic acid, showed early on that the steroid skeleton is constructed from this building block, but not simply through regular head-to-tail coupling reactions: Me Me Me Me Me COOH Me H H H HO 4 A breakthrough came around 1955 with the discovery of mevalonic acid (MVA), which was isolated from concentrated yeast extracts at the end of the beer brewing process. It was also shown that 14C-labelled forms of MVA are efficiently and specifically incorporated into cholesterol. Another important discovery was the isolation and structure determination of squalene from sharks (Squalus), which was also shown to be an efficient biosynthetic intermediate in steroid biosynthesis : Me Me Me Me Me Me COOH H H H HO Me Me H Me Me Me Me OH Me H Me Me Me Me HOOC OH Me Me HO Me Me Me Me In the mean time, all the steps from acetyl-CoA to cholesterol have been established and most of the enzymes involved in the pathway have been isolated and studied. The pathway from acetyl-CoA to MVA, and on to the various classes of terpenes has now been discovered in almost all living organisms, and is known as the mevalonate pathway : O O O O O + + Me SCoA Me SCoA Me SCoA Me SCoA Me OH Me OH Reduction 2x with NADPH + CoASH O SCoA O OH OH OH O (R)-Mevalonic acid O -P- = P O O- 3 ATP Me C5-building blocks Me CO2 + O-P-P O-P-P Me 3 ADP Isopentenyl pyrophosphate Dimethylallyl pyrophosphate (DMAPP) (IPP) The enzyme 3-hydroxy-3-methylglutaryl-CoA synthase catalyzes an Aldol-type reaction that is unusal from a regiochemical viewpoint: Me OH O O O + Me SCoA Me SCoA + CoASH O OH O SCoA 5 Mechanism: O O CoASH S SH SCoA A H O O O O SCoA S S H B O HO Me O H2O + HMGS O HO Me O HO SCoA S SCoA Through crystallographic studies, also with substrates bound at the active site, a good model for the reaction mechanism has been established. The structures have also shown which residues at the active site are most likely involved in catalysis (Vgl PNAS 2004, 101, 16442): A. Acetoacetyl-CoA and Acetyl-Cys, and B. HMG-CoA in the active site In the next step of the mevalonate pathway, the CoAS-thioester group is reduced in a reaction requiring two equivalents of NADPH. The reaction proceeds in two steps (thioester aldehyde alcohol). Many inhibitors of this enzymic reaction have been discovered, and several of these (called statins) are now important pharmaceutical products. The statins (or HMG-CoA reductase inhibitors) form a class of hypolipidemic drugs used to lower cholesterol levels in people with, or at risk of, cardiovascular disease. 6 They lower cholesterol by inhibiting the enzyme HMG-CoA reductase (HMGR), which is the rate-limiting enzyme of the mevalonate pathway of cholesterol synthesis. In the 1970's the Japanese microbiologist Akira Endo first discovered natural products with a powerful inhibitory effect on HMGR in a fermentation broth of Penicillium citrinum, during the search for antimicrobial agents. The first product was named compactin (ML236B or mevastatin). Animal trials showed very good inhibitory effects, however, in a long term toxicity study in dogs toxic effects were observed at higher doses. In 1978, Alfred Alberts and colleagues at Merck Research Laboratories discovered a new natural product in a fermentation broth of Aspergillus terreus, their product showed good HMGR inhibition and they named the product mevinolin, which later became known as lovastatin. HO O HO HO O O HO O O Me O COO O O Me O OH O O H Me SCoA H Me Me SCoA H Me HMG-CoA (Km = 4 µM) Me HO Compactin (IC50 = 23 nM) HO O O Mevinolin O O (Lovastatin) OH OH O OH OH F COOH F N N N H O N Lipitor (Atorvastatin) Fluvastatin (IC50 = 28 nM) Cerivastatin (IC50 = 10 nM) F The so-called "statins" have become important and lucrative drugs for lowering the blood cholesterol level (for a review of their history, importance, mode of action see: Chemie in Unsere Zeit 2010, 44, 344). Lipitor (Atorvastatin (Warner/Lambert, then Pfizer)) was the world's top selling drug in 2009 ($13.2 bill. turnover). The essential structural components of all statins are a dihydroxyheptanoic acid unit and a ring system with different substituents. The statin pharmacophore is a modified hydroxyglutaric acid component, which is structurally similar to the endogenous substrate HMG-CoA and the mevaldyl-CoA intermediate in the enzymic reaction. The statin pharmacophore binds to the same active site as the substrate HMG-CoA and inhibits the HMGR enzyme. It has also been shown that the HMGR is stereoselective and as a result all statins need to have the 3R,5R absolute configuration. Subsequent steps lead to the important C5 building blocks IPP and DMAPP. IPP is isomerized to DMAPP by the enzyme isopentenyl pyrophosphate isomerase: During the past 10 years a very important discovery was made, namely, that in some organisms an alternative pathway exists to DMAPP and IPP. This alternative pathway is found in some microorganisms 7 as well as the plastids of plants and algae, and is called the MEP (2-methyl-D-erythritol-4-phosphate)- pathway (or more simply the non-mevalonate pathway), which is initiated from C5-sugars. The mechanisms of some of the steps in this pathway have not yet been elucidated: Me CO Me O 2 PP HO Me COOH O HO Me CTP i O NADPH HO O PO 2- O P O CMP 3 - CHO TPP HO O OH HO OH OH OH 2- CH2O-PO3 ATP CH2O-PO3 Deoxyxylulose- 5-Phosphate ADP PO2 O Me Me O 2- Me O3PO O 3- PO2 O P2O6 O O P O CMP - HO + HO OH HO OH O H2O H CMP 2e- H+ 2e- Me 3- H2O Me O P2O6 DMAPP Me 3- O P2O6 IPP After the formation of IPP and DMAPP, there exists in all organisms a central route to the universal building blocks needed for mono-, sesqui-, di-, tri and tetra-terpene biosynthesis: C10-Building block Me Me Me Me + Monoterpenes Me O-P-P O-P-P Me O-P-P Geranyl pyrophosphate DMAPP IPP (GPP) C15-Building block Me Me Me Sesquiterpenes Me Me + R O-P-P O-P-P Me O-P-P GPP IPP Farnesyl pyrophosphate (FPP) C20-Building block Me Me Me Me Me Me Diterpenes + O-P-P R O-P-P Me O-P-P IPP Geranylgeranyl pyrophosphate FPP (GGPP) Me Me Me C30-Building block Me R + P-P-O Me O-P-P Me R Me Me Me Me FPP FPP Squalene Triterpenes Me + P-P-O R Steroids O-P-P C40-Building block R Me Tetraterpenes GGPP GGPP The mechanism and stereochemical course of all these steps was investigated by J.