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(photosynthesis) Polysaccharides Glycosides Nucleic Acids phosphoenol pyruvate Shikimate pathway Aromatic Compounds Lignans Shikimate pyruvate acetyl CoA CH3COSCoA aromatic amino acids aliphatic amino acids CITRIC ACID CYCLE Alkaloids Peptides Penicillins Cyclic Peptides CH3COSCoA -O 2CCH2COSCoA CH3COCH2COSCoA CH3COSCoA CH3COSCoA (3) Isoprenoids (terpenes, steroids, carotenoids) mevalonate Polyketides, Fatty Acids Prostaglandins, Macrocyclic Antibiotics (3) Isoprene pathway: Terpenes (3) Mevalonate Terpenes, Steroids -- made from 5-carbon units - C6 compound that loses CO2 to form C5 units phytane (C20) a-pinene (C10) cholesterol (missing 3 C’s) Isoprene Biosynthesis decarboxylates DMAP to yield IPP IPP - Result: two isomeric 5-carbon molecules, IPP + DMAP - Pyrophosphates: high-energy group powers biosynthetic rxns.. “nature’s leaving group” Biosynthesis of Monoterpenes (C10) DMAP IPP Geranyl pyrophosphate (C10) Prenyl transferase + -OR- Cyclase enzymes Farnesyl pyrophosphate (C15) rearranged to form sesquiterpenes (C15) cyclic monoterpenes (C10) Biosynthesis of Monoterpenes (C10) (1) DMAP ionizes to form electrophilic carbocation (2) Nucleophilic attack by IPP forms geranyl-PP (3) Stereospecific loss of HR, forming double bond (4) Geranyl-PP ionizes, rearranges to form a carbocation intermediate - Cyclic monoterpenes then form via enzyme-catalyzed stereospecific rearrangements, functionalizations thujone 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 (+)-carvone = caraway seed (-)-carvone = spearmint (+)-limonene = oranges (-)- limonene = lemons Biosynthesis of Sesquiterpenes (C15) DMAP IPP Prenyl transferase + Farnesyl pyrophosphate (C15) rearranged to form sesquiterpenes (C15) Geranyl pyrophosphate (C10) Biosynthesis of Sesquiterpenes (C15) DMAP IPP Geranyl pyrophosphate (C10) Prenyl transferase + Farnesyl pyrophosphate (C15) rearranged to form sesquiterpenes (C15) - if you introduce a labeled carbon in the precursor, you can see where it ends up in the final natural product Biosynthesis of Sesquiterpenes (C15) DMAP IPP Geranyl pyrophosphate (C10) Prenyl transferase + Farnesyl pyrophosphate (C15) rearranged to form sesquiterpenes (C15) - if you introduce a labeled carbon in the precursor, you can see where it ends up in the final natural product 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 (14C, 3H) - feed cells a precursor with one 14C - use chemical degradation to break natural product apart, see which pieces contain the radioactivity - drawback: sloppy, usually not possible to completely determine biosynthetic route Labeling studies in Biosynthesis 2 Now done by NMR using non-radioactive isotope 13C - common carbon isotope 12C is invisible to NMR - rare 13C has nuclear spin I = n/2, so is detectable by NMR - feed cells a precursor with one position 13C labeled; then see which positions show exaggerated peaks in the carbon NMR spectrum since only 1% of carbons are naturally 13C, the positions that incorporate the label will give much higher peaks Kubanek et al. JOC 1997 13C NMR spectrum of compound 2, after feeding sea slug with [2-13C] mevalonate - increased size of peaks shows label incorporation - allowed researchers to infer biosynthetic pathway for these related C-23 and C-21 terpenes Biosynthesis of Trichodiene (C15) Trichodiene - Precursor (mevalonate) can be labeled at a particular atom with a stable isotope (13C); fate of label then traced in final product Biosynthesis of Trichodiene (C15) Trichodiene - Biosynthesis proceeds via carbocation intermediates, migration of 2 methyl groups and a hydrogen Trichodiene 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) Diterpene (C20) Biosynthesis + Farnesyl pyrophosphate (C15) carbocation intermediate Gerenylgerenyl-PP (C20) Diterpenes Gibberellin Biosynthesis CH3 oxidized, then lost as CO2 Gibberellin A3, a potent C19 plant hormone - found in most plants (though originally isolated from a fungus) Triterpene (C30) Biosynthesis DMAP IPP Geranyl pyrophosphate (C10) Farnesyl pyrophosphate (C15) Squalene (from shark oil) C30 compound: 2 farnesyl’s joined tail-to-tail Triterpene (C30) Biosynthesis Squalene other C30 triterpenes cholesterol (C27) sex hormones vitamin D Cholesterol Biosynthesis - lose 3 methyl groups Vitamin D Biosynthesis - cholesterol from liver is transported to skin - vitamin allows uptake of essential calcium - photochemically converted into vitamin D 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 13C at position 2 - carbon NMR experiments reveal where the label ends up in the completed molecule Higher terpenes (C40) 2 x geranyl geranyl-PP Lycopene - major antioxidant pigment in tomatoes b-carotene - major accessory pigment in photosynthesis The highest terpenes (Cbig #) ~ 1 % of plants can synthesize cis-polyisoprenoids, like rubber H3C Commercially used rubber plants can convert nearly 100% of their mevalonate into rubber (photosynthesis) Polysaccharides Glycosides Nucleic Acids phosphoenol pyruvate Shikimate pathway Aromatic Compounds Lignans Shikimate pyruvate acetyl CoA CH3COSCoA aromatic amino acids aliphatic amino acids CITRIC ACID CYCLE Alkaloids Peptides Penicillins Cyclic Peptides CH3COSCoA -O 2CCH2COSCoA CH3COCH2COSCoA CH3COSCoA CH3COSCoA Isoprenoids (terpenes, steroids, carotenoids) mevalonate Polyketides, Fatty Acids (4) Prostaglandins, Macrocyclic Antibiotics (4) Polyketide Biosynthesis (4) Acetate Polyketides O H3C SCoA Erythromycin A (antibacterial) Avermectin B1 (antihelminthic) (4) Polyketide Biosynthesis (4) Acetate (1) aromatic compounds, usually with meta placement of -OH’s O H3C Polyketides: SCoA (2) non-aromatic macrolides Avermectin B1 6-methyl salicylate (antihelminthic) Polyketide Biosynthesis Compounds constructed by addition of successive 2-carbon units Ketone may or may not be later reduced Options in final structure: OH O -orH3C C H H3C C -or- H3C C Polyketide Biosynthesis Important natural producers of polyketide metabolites: - fungi (= molds) - bacteria, particularly of the family Actinomycetes - sea slugs Polyketide Biosynthesis Actual pathway starts with an acetyl-coA, but then successively uses malonyl coA, which loses CO2 thus adding C2 units - analogous to how mevalonate loses CO2 to add C5 units in terpenoid biosynthesis 1 acetate, 3 malonyl coA’s 4 carbons are labeled in the final product The Lactone Mellein [1,2-13C]-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 Griseofulvin Biosynthesis Start with 14 carbons in linear precursor… …End with 17 ? Griseofulvin Biosynthesis Post-cyclization modifications: (1) addition of methoxy groups (2) halogenation Actinorhodin Biosynthesis Antibiotic from bacterium Streptomyces coelicolor methylated afterwards kalafungin (an antibiotic intermediate) actinorhodin 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 Erythromycin Antibiotic from bacterium Streptomyces erythreus 6-deoxy-erythononlide B Synthesized using propionate, C3 units, instead of 2-carbon acetate units (hence all those methyl groups) Erythromycin cladinose desosamine 2 sugars added last, not part of the polypropionate pathway 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”) Mixed Biosynthesis: Vitamin K isoprene unit Synthesis performed by enteric bacteria in large intestine Part of vitamin is from shikimate pathway, part isoprene Rules for identifying pathways (1) Is there nitrogen? Yes...alkaloid (2) Count carbons a) multiple of 5.... terpene C10 = monoterpene especially if the rings are C15 = sesquiterpene not aromatic C20 = diterpene b) another even number... probably polyketide c) multiple of 3... (w/ lots of methyls) polypropionate 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 Rules for identifying pathways (3) Aromatic... a) hydroxyl groups are meta... b) otherwise... polyketide shikimate You can’t always differentiate these 2 pathways without doing labeling experiments