7. Triterpenes and Sterols

1. 7. Triterpenes and Sterols RA Macahig FM. Dayrit

2. 7.0 Triterpenes & steroids (Dayrit) Introduction • Triterpenes and sterols comprise a large group of distinctive polycyclic terpenes. • Triterpenes are C30 compounds which have four or five fused cyclohexyl rings. • Sterols are C21 to C29 compounds with a characteristic fused ring system of three cyclohexyl rings and one cyclopentyl ring. • Triterpenes and sterols can be isolated in free form, glycosylated, or bound to fatty acids. • Sterols are well known to have important hormonal activity in insects and mammals (including humans).

3. 7.0 Triterpenes & steroids (Dayrit) Squalene • Triterpenes arise from the reductive dimerization of two farnesyldiphosphate chains (2 x C15) which condense in a head-to-head manner to form squalene. • Squalene is the key intermediate in the biosynthetic pathway to the two key triterpene intermediates: cycloartenol (in plants) and lanosterol (in animals). • Important features of squalene: • It does not have a -OPP leaving group. • It is a symmetric molecule.

4. 7.0 Triterpenes & steroids (Dayrit) Biosynthetic mechanism for the head-to-head dimerization of two farnesyl diphosphate chains to produce squalene.

5. 7.0 Triterpenes & steroids (Dayrit) Squalene Squalene itself is found in large quantities in shark liver oil, thus its name (squalus = shark). It is also obtained from vegetable oils, such as rice bran, wheat germ, and olives. It is marketed as a cosmetic and health supplement for protection against oxidative processes which lead to aging, atherosclerosis and other immune diseases. Squalene is found only in the all-trans double-bond configuration. All variations of structures among triterpenes arise from the conformation of folding; there are no geometric isomers of double bonds.

6. 7.0 Triterpenes & steroids (Dayrit) Overview of squalene formation and the biogenetic relationships of triterpenes and sterols.

7. 7.0 Triterpenes & steroids (Dayrit)

8. 7.0 Triterpenes & steroids (Dayrit) TEM of peroxisome showing crystalline and non-crystalline inclusions Early studies on the enzymes involved in cholesterol biosynthesis proposed that all the enzymes involved in the conversion of acetyl-CoA to farnesyl diphosphate (FPP), with the exception of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), are cytosolic or are located on the endoplasmic reticulum. This study shows that FPP is found in peroxisomes. Peroxisomes are ubiquitous organelles in eukaryotic cells that participate in the metabolism of fatty acids and other metabolites. Peroxisomes have a single lipid bilayer membrane that separates their contents from the cytosol (the internal fluid of the cell) and contain membrane proteins critical for various functions.

9. 7.0 Triterpenes & steroids (Dayrit) 2,3-Oxidosqualene cyclase is an integral membrane enzyme that catalyzes the cyclization of squalene epoxide to lanosterol. The solubilized enzyme was purified to homogeneity by fast protein liquid chromatography. The purified enzyme consists of a single subunit that has an apparent molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme obeys saturation kinetics and the apparent Km of (2,3)-oxidosqualene is 15 mM and the apparent kcat/Km is 200 M-1 min-1.

10. 7.0 Triterpenes & steroids (Dayrit) Humans and yeast: squalene synthase (SQS) S. aureus: CrtM Terpene biosynthetic pathways differ in prokaryotes and eukaryotes. The two pathways shown here diverge after presqualene diphosphate. Staphyloxanthin biosynthesis in S. aureus. The NADPH reduction step is absent, resulting in formation of dehydrosqualene, not squalene. Steroid biosynthesis in humans and yeasts passes through squalene. (Liu et al., Science 319, 1391 -1394 (2008))

11. 7.0 Triterpenes & steroids (Dayrit) Overview of cyclization reactions in triterpenes • The triterpene structures are formed from the enzyme-catalyzed cyclization of squalene. The first step involves the activation of the asymmetric squalene by epoxidation of a terminal olefin group producing the chiral intermediate (3S)-2,3-oxidosqualene. • Various modes of cyclization produce 29 major triterpene skeletal types plus about 5 irregular ones. The main sources of variation among the triterpenes are due to: • Conformation of folding (regiochemistry) • Conformation of folding (stereochemistry) • Skeletal rearrangements • Further chemical transformations, such as oxidation, methylation, and glycosylation.

12. 7.0 Triterpenes & steroids (Dayrit) Overview of cyclization reactions in triterpenes 1. The conformation of folding: regiochemistry. (3S)-2,3-oxidosqualene is folded into specific geometries under enzyme control during the cyclization process. These conformations are either chair or boat (c = chair; b = boat). There are four conformational folding patterns: a. To form tetracyclic products, two folding conformations are observed: (c - c - c - b) and (c - b - c - b); b. To form pentacyclic products, the folding conformations are: (c - c - c - c - c) and (c - b - c - c - b); and c. Irregular cyclization leads to other types of triterpenes.

13. 7.0 Triterpenes & steroids (Dayrit) Model for the oxidosqualene cyclase enzyme which controls the transition state conformation of triterpene formation. chair chair boat boat Each folding conformation and product is presumed to require a unique enzyme. A number of cyclases have been isolated and sequences from a number of plants and microorganisms, as well as from pig liver, has been determined.

14. 7.0 Triterpenes & steroids (Dayrit) 2. The conformation of folding: stereochemistry.

15. Triterpenes: an overview

16. Chair - chair - chair - boat conformation of squalene folding to form tetracyclic triterpenes: the Dammaranes.

17. Chair - chair - chair - boat conformation: the Dammaranes.

19. Further modifications on the dammarane skeleton.

20. Further modifications on the dammarane skeleton.

23. Chair-boat-chair-boat conformation: Cycloartenol and Lanosterol.

24. Chair-boat-chair-boat: Cycloartenol and Lanosterol.

25. cycloartenol and lanosterol.

27. 7.0 Triterpenes & steroids (Dayrit) Unusual triterpenes. Ambrein and malabaricol are examples of interrupted cyclization.

28. 7.0 Triterpenes & steroids (Dayrit) Some important triterpenes Corosolic acid (2-hydroxyursolic acid) has been identified as one of the active constituents in the leaves of Banaba (Lagerstroemia speciosa). It has been shown to lower blood sugar in animals. The leaves are very popular as a remedy for diabetes. Maslinic acid (2-hydroxyoleanolic acid), a structurally-related triterpene, has also been identified in the leaves.

29. 7.0 Triterpenes & steroids (Dayrit) Triterpene glycosides • In plants, triterpenes have been isolated in either of two forms: in its free form as an aglycone (that is, without any sugar attached) or as the glycoside (aglycone + sugar). • It is likely that in many cases, the aglycone may have been an artifact of the isolation process; for example, enzymes present in the leaves may hydrolyze the triterpene glycosides, or the extraction process itself may cause hydrolysis. • At any rate, it is reasonable to assume that triterpene glycosides are common constituents of plants and that many triterpenes may exist in the glycoside form in the intact plant.

30. 7.0 Triterpenes & steroids (Dayrit) Ginseng is a very complex mixture of at least 31 ginsenoside triterpenes. Ginseng is used in both traditional and modern forms, especially for maintenance of health in old-age. It is widely used in both Europe and East Asia. In 1994 retail sales in Europe reached about $50M.

31. Triterpenes: summary of cyclization modes: c-c-c-b

32. 7.0 Triterpenes & steroids (Dayrit) Introduction to sterols The sterols make up a large group of compounds which are derived from triterpenes and have the characteristic tetracyclic ring ranging from C27 to C29. The various sterols are differentiated by the following main structural features: 1. Side chain on C17 position 2. Modifications on the A-ring 3. Other modifications 4. Alcohol in the C3 position with the exception of some human hormones. • Notes on the structure: • The C3-hydroxy is always . • The A/B ring fusion can be cis or trans (5-H is  or , respectively).

33. 7.0 Triterpenes & steroids (Dayrit) Introduction to sterols • Sterols are found in all kingdoms. In many organisms, these are synthesized de novo from mevalonic acid  squalene; in some, they are obtained from the diet; in others, they are both synthesized and ingested. • The sterols in the various kingdoms have characteristic structural features, and so are classified according to their respective kingdoms: e.g., mycosterols (fungi), phytosterols (plants), marine sterols (sponges and other invertebrate marine organisms), and zoosterols (animals). • However, it is important to note that the occurrence of specific sterol compounds is not always unique to an organism. For example, although cholesterol is the prototypical zoosterol, it is also produced by some algae and plants. On the other hand, stigmasterol, a typical plant sterol, is not produced by animals.

34. 7.0 Triterpenes & steroids (Dayrit) Introduction to sterols • Sterols perform diverse functions in various organisms. For example, sterols are essential constituents of biological membranes of different organisms. They function as hormones and pheromones in a wide range of organisms ranging from mammals to insects. Synthetic steroids have also been developed to control or mediate in various aspects of human physiology, disease and reproduction. • Sterols are derived from the group of tetracyclic triterpenes using the c-b-c-b folding conformation via a series of transformations starting with lanosterol (in mammals) and cycloartenol (in plants). The primary mammalian sterol is cholesterol (C27) while the primary plant sterol is stigmasterol (C29).

35. 7.0 Triterpenes & steroids (Dayrit) Introduction to sterols • In the literature, the sterols are also classified the following groups, partly based on structure, and partly based on activity: • sterols: steroidal skeleton with a C3 alcohol group • cardiotonic sterols and steroidal saponins: physiologic activity • ecdysones: insect hormones • bile acids: modified steroids in humans • steroidal hormones: steroids which affect physiology

36. From triterpenes to sterols: A. lanosterol to cholesterol in mammals; B. cycloartenol to stigmasterol in plants and fungi.

37. 7.0 Triterpenes & steroids (Dayrit) Major mammalian sterol skeletal structures. (Estrogens and some androgens do not have the C3 alcohol group.)

38. 7.0 Triterpenes & steroids (Dayrit) Major sterol skeletal structures in plants and fungi.

39. 7.0 Triterpenes & steroids (Dayrit) Survey of some important sterols

40. 7.0 Triterpenes & steroids (Dayrit)

41. 7.0 Triterpenes & steroids (Dayrit) Phytosterols • The path from squalene to sterols In plants and fungi: • squalene  cycloartenol  phytosterols and fungal sterols 1. Artificially introduced cycloartenol in plants is incorporated into phytosterols. 2. In plants, cycloartenol is found in large amounts while lanosterol is rare. 3. The conversion of cycloartenol to the phytosterols has also been shown to follow a metabolic grid. Cycloartenol can be funneled into two main pathways: methylation of the intermediate to C28 and C29 phytosterols, or formation of C27 sterols (which includes cholesterol). The most common phytosterol is stigmasterol which is a C29 compound.

42. 7.0 Triterpenes & steroids (Dayrit) Proposed pathway for the conversion of cycloartenol in plants to sitosterol.

43. 7.0 Triterpenes & steroids (Dayrit) Proposed mechanism for the conversion of cycloartenol to phytosterols.

44. 7.0 Triterpenes & steroids (Dayrit) Phytosterols: mixed metabolites

45. 7.0 Triterpenes & steroids (Dayrit) Cardiotonic sterols

46. 7.0 Triterpenes & steroids (Dayrit)

47. Steroidal saponins

48. 7.0 Triterpenes & steroids (Dayrit) Zoosterols The conversion of lanosterol to cholesterol in mammals has been extensively studied using rat liver tissues and in vitro enzyme preparations. It has been found that there is no unique, sequential biosynthetic pathway. That is, the enzymes which catalyze the various transformations are not absolutely specific for a particular substrate. It has also been observed that different enzymes are able to catalyze the same reaction on different substrates. The biosynthesis of cholesterol is best described as a metabolic grid. Lanosterol is converted into cholesterol, the entry compound for all of the steroids in mammals, and are called zoosterols. In the animal liver, lanosterol is converted into cholesterol, while cycloartenol is not metabolized. This sequence of transformations in mammals is as follows: squalene  lanosterol  cholesterol  steroid hormones

49. The pathway for the conversion of lanosterol to cholesterol in mammals is believed to occur through a metabolic grid where there is no single pathway and transformations occur in random order.

50. Lanosterol to cholesterol.