1. Polysaccharides Glycosides Nucleic Acids Shikimate pathway Aromatic Compounds Lignans aromatic amino acids aliphatic amino acids CH 3 COSCoA ( photosynthesis ) phosphoenol pyruvate acetyl CoA CH 3 COSCoA - O 2 CCH 2 COSCoA CH 3 COSCoA Polyketides, Fatty Acids Prostaglandins, Macrocyclic Antibiotics Shikimate CH 3 COCH 2 COSCoA mevalonate Isoprenoids (terpenes, steroids, carotenoids) Alkaloids Peptides Penicillins Cyclic Peptides CITRIC ACID CYCLE (3)

2. (3) Mevalonate Terpenes, Steroids -- made from 5-carbon units  -pinene (C 10 ) cholesterol (missing 3 C’s) phytane (C 20 ) (3) Isoprene pathway: Terpenes - C 6 compound that loses CO 2 to form C 5 units

3. Isoprene Biosynthesis DMAPIPP - Result: two isomeric 5-carbon molecules, IPP + DMAP - Pyrophosphates: high-energy group powers biosynthetic rxns.. “nature’s leaving group” decarboxylates to yield IPP

4. Biosynthesis of Monoterpenes (C 10 ) rearranged to form sesquiterpenes ( C 15 ) Geranyl pyrophosphate (C 10 ) Farnesyl pyrophosphate ( C 15 ) Prenyl transferase + DMAPIPP -OR- Cyclase enzymes cyclic monoterpenes (C 10 )

5. Biosynthesis of Monoterpenes (C 10 ) (1) DMAP ionizes to form electrophilic carbocation (2) Nucleophilic attack by IPP forms geranyl-PP (3) Stereospecific loss of H R, forming double bond (4) Geranyl-PP ionizes, rearranges to form a carbocation intermediate - Cyclic monoterpenes then form via enzyme-catalyzed stereospecific rearrangements, functionalizations

6. thujone

7. (+)-carvone = caraway seed ( - )-carvone = spearmint (+)-limonene = oranges ( - )- limonene = lemons Most cyclic monoterpenes have a distinctive odor - basis of perfume & flavor industries Stereoisomers have different characteristic smells - demonstrates that smell receptors are 3D proteins, i.e. chiral environments that can distinguish enantiomers

8. Biosynthesis of Sesquiterpenes (C 15 ) rearranged to form sesquiterpenes ( C 15 ) Geranyl pyrophosphate (C 10 ) Farnesyl pyrophosphate ( C 15 ) Prenyl transferase + DMAPIPP

9. Biosynthesis of Sesquiterpenes (C 15 ) rearranged to form sesquiterpenes ( C 15 ) Geranyl pyrophosphate (C 10 ) Farnesyl pyrophosphate ( C 15 ) Prenyl transferase + DMAPIPP - if you introduce a labeled carbon in the precursor, you can see where it ends up in the final natural product

10. Biosynthesis of Sesquiterpenes (C 15 ) rearranged to form sesquiterpenes ( C 15 ) Geranyl pyrophosphate (C 10 ) Farnesyl pyrophosphate ( C 15 ) Prenyl transferase + DMAPIPP - if you introduce a labeled carbon in the precursor, you can see where it ends up in the final natural product

11. Labeling studies in Biosynthesis 1 Using isotopically enriched precursors, it is possible to trace the incorporation of building blocks into complex metabolites - follow the label and see which atoms in the natural product end up labeled Originally done with radioactive labels ( 14 C, 3 H) - feed cells a precursor with one 14 C - use chemical degradation to break natural product apart, see which pieces contain the radioactivity - drawback: sloppy, usually not possible to completely determine biosynthetic route

12. Labeling studies in Biosynthesis 2 Now done by NMR using non-radioactive isotope 13 C - common carbon isotope 12 C is invisible to NMR - rare 13 C has nuclear spin I = n/2, so is detectable by NMR - feed cells a precursor with one position 13 C labeled; then see which positions show exaggerated peaks in the carbon NMR spectrum since only 1% of carbons are naturally 13 C, the positions that incorporate the label will give much higher peaks

13. 13 C NMR spectrum of compound 2, after feeding sea slug with [2- 13 C] mevalonate - increased size of peaks shows label incorporation Kubanek et al. JOC 1997

14. - allowed researchers to infer biosynthetic pathway for these related C-23 and C-21 terpenes

15. Trichodiene - Precursor (mevalonate) can be labeled at a particular atom with a stable isotope ( 13 C); fate of label then traced in final product Biosynthesis of Trichodiene (C 15 )

16. Trichodiene - Biosynthesis proceeds via carbocation intermediates, migration of 2 methyl groups and a hydrogen Biosynthesis of Trichodiene (C 15 )

17. Trichothecenes Trichodiene is a key precursor of fungal metabolites called trichothecenes, a major human health problem - fungal toxins are called mycotoxins - often present in moldy grain consumed by cattle or people Winter 1942: thousands of Russians died after eating grains left in the field over winter, due to the war; grain was infected with the trichothecene-producing fungus 1981: USA accused Russia of spraying fungal spores in Vietnam, resulting in foliage contaminated with trichothecenes (false) Trichodiene

18. Diterpene (C 20 ) Biosynthesis Farnesyl pyrophosphate ( C 15 ) + Gerenylgerenyl-PP ( C 20 ) carbocation intermediate Diterpenes

19. Gibberellin Biosynthesis CH 3 oxidized, then lost as CO 2 Gibberellin A3, a potent C 19 plant hormone - found in most plants (though originally isolated from a fungus)

20. DMAPIPP Squalene (from shark oil) C 30 compound: 2 farnesyl’s joined tail-to-tail Geranyl pyrophosphate ( C 10 ) Farnesyl pyrophosphate ( C 15 ) Triterpene (C 30 ) Biosynthesis

21. Squalene other C 30 triterpenes cholesterol (C 27 ) sex hormones vitamin D

22. Cholesterol Biosynthesis - lose 3 methyl groups

23. Vitamin D Biosynthesis - cholesterol from liver is transported to skin - photochemically converted into vitamin D - vitamin allows uptake of essential calcium

24. Notes on Terpenes (1) Oxidation reactions are carried out by the enzyme cytochrome P450 - activate oxygen to introduce -OH, carboxyl groups - allow removal of C’s through decarboxylation (2) Triterpenes form flexible rings (chair, boat conformations) with many chiral centers {rings usually not aromatic} - provides a huge number of potential 3D structures - high degree of biological activity (3) Pathways can be elucidated using labeled precursors, such as mevalonate with a 13 C at position 2 - carbon NMR experiments reveal where the label ends up in the completed molecule

25. Higher terpenes (C 40 ) Lycopene 2 x geranyl geranyl-PP - major antioxidant pigment in tomatoes  -carotene - major accessory pigment in photosynthesis

26. The highest terpenes (C big # ) ~ 1 % of plants can synthesize cis-polyisoprenoids, like rubber H3CH3C Commercially used rubber plants can convert nearly 100% of their mevalonate into rubber

27. Polysaccharides Glycosides Nucleic Acids Shikimate pathway Aromatic Compounds Lignans aromatic amino acids aliphatic amino acids CH 3 COSCoA ( photosynthesis ) phosphoenol pyruvate acetyl CoA CH 3 COSCoA - O 2 CCH 2 COSCoA CH 3 COSCoA Polyketides, Fatty Acids Prostaglandins, Macrocyclic Antibiotics Shikimate CH 3 COCH 2 COSCoA mevalonate Isoprenoids (terpenes, steroids, carotenoids) Alkaloids Peptides Penicillins Cyclic Peptides CITRIC ACID CYCLE (4)

28. (4) Acetate Polyketides H3CH3CSCoA O Erythromycin A (antibacterial) Avermectin B 1 (antihelminthic) (4) Polyketide Biosynthesis

29. (4) Acetate Polyketides: (1) aromatic compounds, usually with meta placement of -OH’s (2) non-aromatic macrolides H3CH3CSCoA O Avermectin B 1 (antihelminthic) (4) Polyketide Biosynthesis 6-methyl salicylate

30. Polyketide Biosynthesis Compounds constructed by addition of successive 2-carbon units Ketone may or may not be later reduced Options in final structure: H3CH3CC O H3CH3CC OH H H3CH3CC -or-

31. Polyketide Biosynthesis Important natural producers of polyketide metabolites: - fungi (= molds) - bacteria, particularly of the family Actinomycetes - sea slugs

32. Polyketide Biosynthesis Actual pathway starts with an acetyl-coA, but then successively uses malonyl coA, which loses CO 2 thus adding C 2 units - analogous to how mevalonate loses CO 2 to add C 5 units in terpenoid biosynthesis 1 acetate, 3 malonyl coA’s 4 carbons are labeled in the final product

33. The Lactone Mellein [1,2- 13 C]-acetate From the fungus Aspergillus Growing polyketide chains are held bound to the biosynthetic enzyme, passed from one active site to the next Different active sites carry out the various cyclizations + reductions The final site is a thioesterase, which cleaves the connection, setting the compound loose

34. Griseofulvin Biosynthesis Start with 14 carbons in linear precursor… …End with 17 ?

35. Griseofulvin Biosynthesis Post-cyclization modifications: (1) addition of methoxy groups (2) halogenation

36. Actinorhodin Biosynthesis actinorhodin Antibiotic from bacterium Streptomyces coelicolor kalafungin (an antibiotic intermediate) methylated afterwards

37. Aflatoxin Isolated in 1960 after thousands of turkeys died of liver failure from eating moldy peanut meal; culprit was Aspergillus mold Now evidence that in parts of the world where peanuts are a dietary staple, much liver disease is due to persistent aflatoxin consumption

38. Erythromycin Synthesized using propionate, C 3 units, instead of 2-carbon acetate units (hence all those methyl groups) Antibiotic from bacterium Streptomyces erythreus 6-deoxy-erythononlide B

39. Erythromycin 2 sugars added last, not part of the polypropionate pathway cladinose desosamine

40. Erythromycin Biosynthetic genes for erythromycin have been cloned: - made by 3 large, polyfunctional proteins called modular polyketide synthases - proteins have a series of similar active sites that carry out each successive step in the biosynthesis (hence “modular”)

41. Mixed Biosynthesis: Vitamin K Synthesis performed by enteric bacteria in large intestine Part of vitamin is from shikimate pathway, part isoprene isoprene unit

42. Rules for identifying pathways (1) Is there nitrogen? Yes...alkaloid (2) Count carbons a) multiple of 5....terpene C 10 = monoterpene C 15 = sesquiterpene C 20 = diterpene b) another even number...probably polyketide c) multiple of 3...polypropionate (w/ lots of methyls) especially if the rings are not aromatic

43. Rules for identifying pathways Hints for carbon-counting: 1) non-skeleton carbons don’t count - Those attached through oxygen (esters, ethers) are usually (but not always) added after the main pathway - For example, in R-OMe, the methoxy carbon would not be included in your count (secondary modification) 2) sugars do not count; they are often tacked onto shikimate, polyketide metabolites after the synthesis is complete

44. Rules for identifying pathways (3) Aromatic... a) hydroxyl groups are meta...polyketide b) otherwise...shikimate You can’t always differentiate these 2 pathways without doing labeling experiments