Natural products Primary and secondary metabolism Primary metabolism

1. Natural products Primary and secondary metabolism Primary metabolism -All organisms need to transform and interconvert a vast number of organic compounds to enable them to live, grow and reproduce. -They need to provide themselves with energy in the form of ATP, and a supply of building blocks to construct their own tissues. -An integrated network of enzyme-mediated and carefully regulated chemical reactions is used for this purpose (metabolic pathways).

2. -Some of crucially important molecules of life are carbohydrates, proteins, fats, and nucleic acids. Apart from fats, these are polymeric materials. • -Carbohydrates are composed of sugar units, whilst proteins are made up from amino acids, and nucleic acids are based on nucleotides. • -Organisms vary widely in their capacity to synthesis and transform chemicals. • -For instance, plants are very efficient at synthesizing organic compounds via photosynthesis from inorganic materials found in the environment, whilst other organisms such as animals and microorganisms rely on obtaining their raw materials in their diet, e.g. by consuming plants.

3. -Thus, many of the metabolic pathways are concerned with degrading materials taken in as food, whilst other metabolic pathways are required to synthesize specialized molecules from the basic compounds so obtained. • -Despite the extremely varied characteristics of living organisms, the pathways for generally modifying and synthesizing carbohydrates, proteins, fats and nucleic acids are found to be essentially the same in all organisms, apart from minor variations. • -These processes demonstrate the fundamental unity of all living matter, and are collectively described as primary metabolism, with the compounds involved in these pathways being termed primary metabolites.

4. - Thus degradation of carbohydrates and sugars generally proceeds via the well characterized pathways known as glycolysis and the Krebs cycle, which release energy from the organic compounds by the oxidation reactions. • -Oxidation of fatty acids from fats by the sequence of β-oxidation also provides energy. • -Protein taken in via diet provide amino acids.

5. Primary metabolism comprises the chemical processes that every plant must carry out every day in order to survive and reproduce its line. • PRIMARY METABOLISM Photosynthesis Glycolysis Citric Acid Cycle Synthesis of amino acids Transamination Synthesis of proteins and enzymes Synthesis of coenzymes Synthesis of structural materials Duplication of genetic material Reproduction of cells (growth) Absorption of nutrients

6. Secondary metabolism • -In contrast to these primary metabolic pathways, which synthesize, degrade, and generally interconvert compounds commonly encountered in all organisms, there also exists an area of metabolism concerned with compounds which have a much more limited distribution in nature. • -Such compounds, called Secondary metabolites, are found in only specific organisms, • -Secondary metabolites are not necessarily produced under all conditions, and in the vast majority of cases the function of these compounds and their benefits to the organism is not yet known.

7. -Some are undoubtedly produced for easily appreciated reasons, e.g. toxic materials providing defense against predators, as volatile attractants towards the same or other species, or as colouring agents to attract or warn other species, but it is logical to assume that all do plays some vital role for the well-being of the producer. • -It is this area of secondary metabolites that provides most of the pharmacologically active nature products. • -It is thus fairly obvious that the human diet could be both unpalatable and remarkably dangerous if all plants, animal and fungi produced the same range of compounds.

8. - Secondary metabolism comprises the chemical processes that are unique to a given plant, and are not universal. - Secondary metabolism is the chemistry that leads to the formation of a naturalproduct. -Sometimes portions of this chemistry are common to a number of different plants or plant families, but the actual chemical produced (natural product) is usually different in one plant than in another. -Common chemical precursors can lead to different results. - Secondary metabolites (in most cases) do not appear to be necessary to the survival of the plant, but they may give it a competitive advantage.

9. A TYPICAL PLANT hn Glycolysis ENERGY Photosynthesis (daytime) CO2 Respiration H2O (nighttime) O2 N2 bacteria “N” TRACE METALS Na, Ca, K, Mg Fe, Cu, Co, Mo NO2-/NO3-/NH4+ H2O

10. CO2 + H2O PRIMARY METABOLISM hn Photosynthesis Glucose Carbohydrates SECONDARY METABOLISM SECONDARY METABOLISM GLYCOLYS IS Building Blocks Phenyl- propanoids Amno Acids Fatty Acids Lipids Flavonoids Proteins synthesis Alkaloids Acetyl CoA enzymes Acetogenins Terpenes Steroids regulation Nucleic Acids Citric Acid Cycle reproduction RNA DNA CO2 + H2O + ATP

11. hn CO2 polyketides acetogenins lipids fatty acids phenylpropanes citric acid cycle alkaloids Glucose (6 carbons) photosynthesis starch n glycolysis erythrose- 4-phosphate phosphoenol pyruvate (PEP) (3 carbons) shikimic acid anthanilic acid acetyl- coenzymeA (2 carbons) mevalonic acid lysine ornithine oxalo- acetate phenylalanine tyrosine energy (ATP) + CO2 + H2O tryptophan terpenes steroids carotenoids NH3 nicotinic acid aspartic acid glutamic acid

12. The buildings blocks • -The building blocks for secondary metabolites are derived from primary metabolism. • - The most important building blocks employed in the biosynthesis of secondary metabolites are derived from the intermediates acetyl coenzyme A (Acetyl-CoA), shikmic acid, mevalonic acid, and 1-deoxyxylulose 5-phosphate. • - Acetyl-CoA is formed by oxidative decarboxylation of pyruvic acid and β-oxidation of fatty acids. It used for synthesis of phenols, prostaglandins, and macrolide antibiotics.

13. -Shikimic acid is produced from a combination of phosphoenolpyruvate and erthro 4-phosphate. The shikimate pathway leads to a variety of phenols, cinnamic acid deravatives, lignans, and alkaloids. • -Mevalonic acid is formed from three molecules of acetyl-CoA. • - Deoxyxylulose phosphate arises from a combination of pyruvic acid and glyceraldehyde 3-phosphate.

14. -The mevalonate and deoxyxylulose phosphate pathways are together responsible for the biosynthesis of vast array of terpenoid and steroid metabolites. • - Some amino acids (phenylalanine, tyrosine, tryptophane, ornithine, and lysine) are employed in natural product synthesis, peptides, proteins, alkaloids, and many antibiotics.

15. The strucrural features of the building blocks: • 1- C1: The simplest of the building blocks is composed of a single carbon atom, usually in the form of a methyl group, and most frequently it is attached to oxygen or nitrogen -X-CH3 (X= O,N), it derived from methionine. • 2- C2: A two carbon unit may be supplied by acetyl-CoA (C=C). It could be a simple acetyl group forms part of ester.

16. 3- C5: The branched-chain C5 'isoprene' is formed from mevalonate or deoxyxylulose phosphate. Mevalonate itself is the product from three acetyl-CoA molecules, but only five of six carbons are used, the carboxylic group being lost.

17. head tail isoprene unit isoprene

18. 4- C6C3: This refers to a phenylpropyl unit and is obtained from L-phenylalanine or L-tyrosine, two of the shikimate-derived aromatic amino acids. This requires loss of amino groups. Sometimes the side chain is cleaved, removing one or two carbons (C6C2 or C6C1). • 5- C6C2N: derived from L-phenylalanine and L-tyrosine. This requires loss of carboxyl groups. • 6- Indol.C2N: derived from tryptophan. This requires loss of carboxyl groups.

19. tyrosine phenylalanine Phenylpropyl C6C3 or C6C1

20. Tryptophane Indol.C2N

21. 7- C4N; derived from L-ornithine. The carboxylic and α-amino nitrogen groups are lost. • 8- C5N: derived from lysine. The carboxylic and amino nitrogen groups are lost. • These eight building blocks will form the basis of many of the natural product structures.

22. C4N & C5N

23. The construction mechanisms • Natural product molecules are biosynthesized by a sequence of reactions which are catalysed by enzymes. Some of the important reactions frequently encountered are now described; • 1- Alkylation reactions: • 2- Wagner-Meerwein rearrangements: • 3- Aldol and claisen reactions: • 4- Schiff base formation and the Mannich reaction:

24. 5- Transamination: is the exchange of the amino group from an amino acid to a keto acid. The couple glutamic acid/oxoglutaric acid are the usual donor/acceptor molecules for the amino group. 6-Dehydrogenases: remove two hydrogen atoms from the substrate, passing them to a suitable coenzyme acceptor. The coenzyme system involved can generally be related to the functional group being oxidized in the substrate. Thus if the oxidation process is CH-OH →C=O the NAD or NADP tends to be utilized as hydrogen acceptor. One hydrogen from the substrate (that bonded to carbon) is transferred as hydride (H-) to the coenzyme, and the other, as proton, is passed to the medium.

25. 7- Oxidases: also remove hydrogen from a substrate, but pass these atoms to molecular oxygen or to hydrogen peroxide, in both cases forming water. Oxidases using hydrogen peroxide are termed peroxidases.

26. 8- Monooxygenases : introduce one atom from molecular oxygen into substrate. The second oxygen atom from O2 is reduced to water by an appropriate hydrogen donor e.g. NADH, NADPH, or ascorbic acid (vitamin C). Cytochrome P-450-dependent mono-oxygenases are important examples of these enzymes. These are involved in biological hydroxylations either in biosynthesis , or in detoxification and metabolism of foreign compounds such as drugs. 9- Dioxygenases: introduce both atom molecular oxygen into the sustrate, and are frequently involved in the cleavage of bonds, including aromatic rings.

27. 10- Amine oxidases: These include monoamine oxidases and diamine oxidases. Monoamine oxidases utilize FAD, O2 lead to aldehyde and NH3 (RCH2NH2 +O2+FAD→ RCHO +NH3). Diamine oxidases require diamine substrateb, oxidize at one amino group (H2N-CHO).

28. 11- Bayer-Villiger oxidations: The chemical oxidation of ketones by peracid, the Bayer-Villiger oxidation, yields an ester, and the process is known to involve migration of an alkyl group from the keton. 12- Phenolic oxidative coupling: Many natural products are produced by the coupling of two or more phenolic systems. The reactions can be brought about by oxidase enzyme, including peroxidase system, known to be radical generators.

29. 13- Glycosylation reactions. The widespread occurrence of glycoside and polysaccharides requires process for attaching sugar units to suitable atom of an aglycone to give a glycoside, or to another sugar giving a polysaccharide. Linkages tend to be through oxygen. The agent for glycosylation is a uridine diphoshosugar as UDP-glucose. This is synthesized from glucose 1-phosphate and UTP. The hydrolysis of glycosides is achieved by specific hydrolytic enzymes, e.g. β-glucosidase for β-glucosides and β-galactosidase for β-galactosides.

30. Terpenoids • Terpenoids are secondary metabolites synthesized by plants, marine organisms and fungi by head to tail joining isoprene units (5 carbon atoms). They are also found to occur in rocks, fossils and animal kingdom.

31. Terpenoids find wide applications in industry. For example, linalool along with phenyl-ethyl alcohol is used in perfumery. • Citral is used as a mosquito repellant and the starting material for the synthesis of vitamin A, menthol and artemisinin in pharmaceutical industries. • Farnesol and juvabione are insect juvenile hormones whereas ecdysones are insect anti-moulting hormones. • Gibberellic acids and brassinolides are the plant growth regulators. Salannin and azadirachtins are insect antifeedant and growth inhibitors.

32. Taxol and cucurbitacins are anti-tumor compounds. Forskolin is a unique adenylate cyclase stimulator, which displays a variety of biological activities including blood pressure lowering, positive inotropic, hypolipidemic, antiglucoma. • Artemisinin is a sesquiterpene peroxide with potent antimalarial activity. • Panaxadiol and panaxatriols are immunostimulants. Natural rubber is a polymer of isoprene. Moreover insects use many terpenoid-derived molecules for their communications.

33. Classification • Terpenoid are classified according to the number of isoprene units involved in their biosynthesis. The terpenoid skeletons occur as open chain as well as in various cyclised forms. • 1 unit of isoprene C5 (meroterpenoids) • 2 units of isoprene C10 (monoterpenoids) • 3 units of isoprene C15 (sesquiterpenoids) • 4 units of isoprene C20 (diterpenoids) • 5 units of isoprene C25 (sesterterpenoids) • 6 units of isoprene C30 (triterpenoids) • 8 units of isoprene C40 (carotenoids) • Many of isoprene (natural rubber)

34. Biosynthesis Biosynthesis of terpenoids consists of the following steps: 1- Photosynthesis converts 6 molecules of carbon dioxide and molecules of oxygen to the glucose molecule, which breaks down through glycolysis to pyruvate. 2- Pyruvate is further converted to acetyl-CoA and two molecules of acetyl-CoA couple each other to form acetoacetyl-CoA.

35. 3- Acetoacetyl-CoA reacts with another molecule of acetyl-CoA to produced hydroxymethylglutaric acid which turn to mevalonic acid (MVA) (key-intermediate). 4- MVA is phosphorylated by ATP to give mevalonic acid -5-diphosphate. 5- MVA-5-diphosphate undergoes decarboxylation with the simulantaneous elimination of pyrophosphate to isoprene unit, isopenetenyl diphosphate (IDP).

36. 6- IDP is isomerised by the catalytic action of sulphhydryl enzyme IDP-isomerase to dimethylallyl diphosphate (DMADP). 7- Condensation of DMADP and IDP catalyzed by the enzyme geranyl transferase results in the formation of geranylpyrophosphate (C10) and further addition of IDP units afford farnesyl pyrophosphate (C15) and geranyl-geranyl pyrophosphate (C20), which are precursors of sesquiterpenes and diterpenes, respectively.

37. 8- Two molecules of farnsyl pyrophosphates couple with each other through the cyclopropane intermediate and by the transfer of hydrogen from NADPH to furnish squalene. 9- Squalene is the precursor of all triterpenoids (C30). 10- Condensation of geranyl-geranyl pyrophosphates with IDP leads to C25 skeletone, which is precursor of sesterterpenoids.

38. 11- Two molecules of geranyl-geranyl pyrophosphates couple with each other to afford carotenoids (C-40-isoprenoids). 12- Cyclization in terpenoids: using The complex terpene cyclase reaction.

41. MEVALONIC ACID PATHWAY FORMATION OF MEVALONIC ACID FROM ACETYL UNITS NADPH 3 acetylCoA : : .. - NADPH H2O mevalonic acid (continued next slide)

42. MEVALONIC ACID PATHWAY The isopentenyl and 3,3-dimethylallyl pyrophosphate intermediates ADP AMP :B-Enz 2 ATP mevalonic acid These five-carbon intermediates are responsible for the formation of all the terpenes. Enz-B: H+ IPP DMAPP 3,3-dimethylallyl pyrophosphate isopentenyl pyrophosphate

43. TERPENES The Czech chemist Leopold Ruzicka ( born 1887) showed that many compounds found in nature were formed from multiples of five carbons arranged in the same pattern as an isoprene molecule (obtained by hydrolysis of natural rubber). natural rubber head . D C tail C C C C isoprene isoprene unit He called these compounds “terpenes”.

44. JOINING ISOPRENE UNITS individual isoprene units join head-to-tail Head-to-Tail an extra bond Tail-to-Tail The terms head-to-tail and tail-to-tail are often used to describe how the isoprene units are joined. larger terpenoid units dimerize tail-to-tail ….. explained later

45. SESQUITERPENES guaiazulene geranium oil caryophyllene oil of cloves

46. TRITERPENES TAIL-TO-TAIL ambrein ambergis

47. TETRATERPENE head-to-tail tail-to-tail head-to-tail b-carotene carrots

48. HOW THE TERPENES ARE FORMED

49. CONCATENATION OF C5 (ISOPRENE) UNITS DMAPP IPP C5 C10 HEAD :B C5 isopentenyl-PP 3,3-dimethylallyl-PP C10 TAIL geranyl-PP C15 C20 C15 farnesyl-PP C20 again geranyl-geranyl-PP EACH NEW UNIT IS JOINED HEAD-TO-TAIL

50. C30 AND C40 UNITS ARE FORMED DIFFERENTLY Number Isoprene Class of Carbons Units Origin HEMITERPENES C5 1 IPP or DMAPP MONOTERPENES C10 2 geranyl-PP x2 SESQUITERPENES C15 3 farnesyl-PP x2 DITERPENES C20 4 geranyl-geranyl-PP SESTERTERPENE C25 5 uncommon TRITERPENES C30 6 2 x (farnesyl-PP) C35 7 uncommon TETRATERPENES C40 8 2 x (geranyl-geranyl-PP)