Lecture 4-6.docx

LECTURE 4 SESQUITERPENOIDS Sesquiterpenoids – made up of 3 isoprene units (C15) ⇒ largest & most diverse group of terpenoids >100 basic skeletons → able to make many compounds >1000 known compounds derived from FPP ⇒ categorized into basic groups with many subgroups Acyclic Monocyc...

LECTURE 4 SESQUITERPENOIDS Sesquiterpenoids – made up of 3 isoprene units (C15) ⇒ largest & most diverse group of terpenoids >100 basic skeletons → able to make many compounds >1000 known compounds derived from FPP ⇒ categorized into basic groups with many subgroups Acyclic Monocyclic Bicyclic Mixed/irregular ⇒ properties include Hydrophobic Not very volatile (unlike most monoterpenoids) Most are oxygenated ⇒ used for: Phytoalexins – natural herbicide (antimicrobial or anti-oxidant) plant & animal hormones fungal antibiotics → how fungi kill bacteria anti-tumor agents → natural anticancer drugs IPP + GPP (DMAPP + IPP) → Farnesyl-PP (FPP) ⇒ head to tail condensation via prenyl transferase (catalyst) Farnesyl-PP (FPP) – exists as the 2-trans, 6-trans, & 2-cis,6-trans isomers ⇒ intermediate synthesis of terpenes & terpenoids E.g., sterols & caretonids ⇒ isomerization occurs before cyclization → can cyclize to many compounds Acyclic – relatively few sesquiterpenoid acyclics ⇒ farnesol – alcohol derivative of FPP that gives rise to majority of sesqui-acyclics Important starting compound for organic synthesis Colorless Doubles to 30c squalene → steroids ⇒ Farnesol is used as an additive in perfume Ex. enhances scent of lilac perfumes Ex. acts as co-solvent to regulate volatility of scent (more farnesol = less volatile) Abscisic acid (ABA) – mono-cyclic plant hormone that is biosynthesized within the chloroplast (most studied sesquiterp) ⇒ +ABA is a natural compound Enantiomer is active ⇒ commercial ABA is racemic mix ⇒ xanthoxin – intermediate in ABA pathway that is derived from xanthophyll (carotenoid) ⇒ functions include: Potent plant hormone Growth inhibition (seed dormancy) Leaf abscission (senescence) Stomatal regulation (controls water retention in leaves) Drought tolerance → [ABA] up to 10-20x in response to water limited environment Upregulation of ABA is fast → seconds to minutes Juvenile hormone – key sequiterp that is produced by corpora allata (endocrine gland) as found in insect (silkworms) ⇒ 3 forms of JH at least C18 → R1+R2 = CH3 (neotenin) C17 → R1 = CH3, R2 = H C16 → R1+R2 = H ⇒ prevents metamorphosis → maintains larval state High [JH] = cuticle remains larval type Low [JH] = pupal cuticle forms Absence of JH = adult forms ⇒ can be commercially exploited Regulate insect-based food productions (e.g., honey bees) ⇒ biosynthesis of JH depends on which carbon C16 JH = product of 3 MVA (i.e., GPP + IPP with Me in ester from SAM) C17 JH = product of 2 MVA (gives GPP) + 6C unit from homo-MVA (7C) + 1C from SAM C18 JH = product of 1 MVA + 2 6C units from 2 homo-MVA + 1C from SAM Note – at least 3 units of isoprene no matter what → extra unit is from how they source other units of the compound Juvabione – effective JH analogue that is isolated from balsam fir (agonist) ⇒ insects eat trees that have high juvabione → fail to mature (lethality) Impacts larval development Only impacts certain insects impacted → selective toxicity Cyclic sesquiterp – many types of compounds due to hydride shift & cyclization ⇒ carbon ion makes many common intermediates from the loss of OPP Y-bisabolene (not source of ABA) Picotaxane – present in plants (kills cattle) Humulene – present in plants (antibacterial) Farnesane – example of natural sesqui terp ⇒ e.g., acyclic sesquiterp griffithoside from leaves of Fraxinus griffithii Himalayan ash or evergreen ash Grown in Australia as menthol plant → invasive species LECTURE 5 BASIC DIVERSITY OF TERPENOIDS Chemistry → skeleton ⇒ single structure contributing to multiple compounds in a pathway Chemical reactions Plant features Stress & environmental factors Genetics Combination of above factors FACTORS ENABLING DIVERSITY Arms race – organism constantly using multiple characteristics in attempt to come up with new compounds Role in primary metabolism – found in highly conserved processes ⇒ hormones, electron transfer chain, protein modification, membrane structure, antioxidants, etc. Evolution – heavily involved in diversity of terpenoids even prior to emergence of plants Genes – emergence of new genes or existing genes undergoing mutation that encode for new metabolites Ecological interactions – modulates ecological interactions with the environment ⇒ defense & mutualism (e.g., with pollinators) ECONOMIC IMPACT OF DIVERSITY Economic impact of terpenoids ⇒ one of the first classes to be researched ⇒ several are commercially important Pharmaceuticals, consumer products (rubber), etc. Molecular mechanisms support diversity of terps Role of diversity of terps in plant adaption to different environments & situations ⇒ ancient use for disease treatment in middle east ⇒ other uses for thousands of years Ornaments & religious artifacts using amber (terp plant exudate) Scents (e.g., myrrh & frankincense) Pitch (pine or spruce) → for waterproofing structures such as boats & ships (residue terp following evaporation of volatile terpenes) Source of nutirents → health benefits ⇒ e.g., β-carotene → vitamin A Antioxidant, good for eye sight Contributes significantly to food colour & flavour ⇒ herbs, spices, wine, etc. Bixin Lycopene Astaxanthin Zingiberene – responsible for taste & smell of ginger Nootkatone Have antimicrobial & insecticidal properties that allow for ⇒ food preservation ⇒ ancient use in treatments ⇒ mouthwash ⇒ cough management ⇒ disinfectant ⇒ insect repellent Cell toxicity → can act as anticancer agent ⇒ taxol ⇒ vinblastine Hormonal effects ⇒ diosgenin – sterols found in Mexican yam ⇒ progesterone – for birth control pills ⇒ steroids – in medicine Neuro-effects (toxic & useful) ⇒ cardenolides – e.g., digitoxigenin (cardiac toxicity & treatment) ⇒ tetrahydrocannabinol (THC) – e.g., cannabis (marijuana) ⇒ salvinorin A – salvia divinorum Commercial usage ⇒ can be found in significant amount of trees Results in forest fires due to volatility ⇒ conifers in northern hemisphere ⇒ eucalyptus in Australia ⇒ exploited for fuel using biotechnology Biotech also used in new frug commercial production → Taxol, vinblastine, artemisinin (malaria treatment) MOLECULAR MECHANISMS ENABILING DIVERISTY Condensation reactions – combination/joining od chains allows for compound to modify existing substrates from basic structure ⇒ two molecules combine to form a single molecule Results in removal of a small molecule (usually water) ⇒ reactions occur as: Head to tail → used to be referred to as regular Head-to-Head → used to be referred to as irregular Head to Middle (irregular) → e.g., lavandulol & chrysanthemum acid ⇒ utilize prenyl diphosphates instead of free prenyls → C10 (mono), C15 (sesqui), C20 (di) etc. Head to tail can occur in two ways → trans & cis-prenyl diphosphates Head-to-head of 2 trans sesquiterp diphosphates → C30 (tri) e.g., sterols Head-to-Head of 2 trans diterpenic diphosphates → C40 (tetra) carotenoids Note - α & β-carotene are abundant in photosynthetic tissues while abscisic acid & strigolactones are degradation product of carotenoids Yields – several hormones & sterols ⇒ hormones including Gibberellins Brassinosteroids ⇒ sterols including Stigmasterol Sitosterol Campesterol Unique skeleton – capable of elongation ⇒ cytokinin → 1 unit ⇒ phylloquinone, tocopherols & chlorophylls → C20 attached (two monoterpenoid) ⇒ polyprenols → C25 or more May be free or attachef to form other compounds such as ubiquinones & plastoquinone ⇒ rubber → reaches hundreds of C5 units attached to trans polyprenol Why is rubber so complex? It has a unique skeleoton dur to condensation reaction Specialized metabolites – variety of plant specific metabolites in local environment contributes to diversity of terps ⇒ several thousand or more metabolites exist ⇒ monoterp, sesquiterp, diterp, & triterps Located in flowers & leaves E.g., menthol, artemisinin, taxol ⇒ triterp, & caretonids E.g., steroidal alkaloids, cardenolids & bixin ⇒ others such as strychnine & vinblastine which are derived from smaller terps (monoterp alkaloids) derived partly from secologanin Belongs to iridoid monoterpenes ⇒ addition of prenyl units sometimes following by changes/modification E.g., humulone, THC, & psoralen Structural diversity – reactions are catalyzed by terpene synthases (TPSs) ⇒ TPS uses prenyl diphosphates as precursors → basic terpene skeleton ⇒ TPS product several products from one substrate Due to stochastic charge migrations in carbocation intermediate that occurs in the active enzyme Single amino acid change in TPS → production of multiple different terps ⇒ TPS genes occur as a family of 30-100 genes per genome Enables new terps to appear through mutation & selection ⇒ terpene skeletons are prone to modification reactions by enzymes such as Oxidase, methyltransferase, acyltransferase & prenyltransferase Carbocations – ion with positively charged carbon ⇒ may have more than one positive charge on same or different carbon atoms ⇒ normally go through rearrangement reactions → stability This is important as it leads to multiple structures Can also achieve stability through resonance by C=C next to ionized carbon Rearrangement reactions – carbon skeleton is changed to form structural isomer of original molecule ROLE OF TERP DIVERSITY IN ADAPTATION Hydrophobicity – terps are highly hydrophobic ⇒ localized in membrane or close to it through prenyl group ⇒ examples include ubiquinone & plastoquinone Electron transport chain relies on prenyl chain-mediated membrane association Concentration – [triterpene sterols] in membrane affects fluidity of plant membranes ⇒ terps can have varying effects at different concentrations ⇒ high diversity of structures → use of terpenes as hormones recognized by specific receptors E.g., gibberllins, cytokinins, auxins, brassinolides, & strigolactones ⇒ plant may have multiple forms of hormone due to different modification reactions over time Oxidation, reduction, methylation, esterification & other reactions to which most terpenes are susceptible as described above ⇒ modification reactions help refine the effect of hormone on receptor Defense – one of the primary roles of plant terps ⇒ as toxins, repellents or attracting predators/enemies of herbivores Note not all roles of plant terps have been elucidated Counter-defense – evolutionary “arms race” provides counter defense between plants & specialized herbivores ⇒ through addition of a more complex compound or modification of existing molecule by newly evolved methyl & acyl transferases or oxidative enzymes E.g., diversity of benzoxazinoids in maize & glucosinolates in mustards ⇒ arms race vs defense → form of counter defense due to resilience or other effects Making new or changing compounds to overwhelm a pathogen Mutualism – may start off as toxic & selectively identify organisms that are beneficial to the plant ⇒ i.e., fruit pollution LECTURE 6 DITERPENOIDS Diterpenoids – C20 skeleton from geranyl-geranyl-PP (core) ⇒ formed through head to tail condensation of IPP + FPP Some cases formed through FPP + DMAPP ⇒ mostly cyclic with a few acyclics ⇒ involved in primary & secondary plant metabolism 1° - gibberellin (GA) phytohormones & phytol side chain of chlorophyll 2° - phytoalexins (antibiotic by plant) have been shown to confer resistance against pests or pathogens Several thousand diterps known due to modification & cyclization of GGPP ⇒ not as volatile as sesquiterp E.g., will stay in resin as turpentine is distilled away ⇒ difficult to separate & analyze ⇒ properties include Structure composed of 1 to 5 ring compounds Most are oxygenated ⇒ usages include Drugs → anti-cancer drug Taxol (paclitaxel) Scents Flavours Groups of cyclics → bi-, poly-, & macro-cyclic ⇒ primary bicyclic skeleton – resins of many plants labdane ⇒ polycyclics – in resins pimarane beyerene ⇒ macrocyclic – limited distribution lathyrane phytol – key acyclic that is estrified to porphyrin ring of cholorphyll ⇒ chlorphyll is one of the most abundant terps phytol confers lipid solubility to cholorphyll ⇒ presence in ancient sediments & petroleum points to evolutionary early presence of terps & chlorophyll ⇒ precursor to Vitamin E (α-tocopherol) & K (phylloquinone) phorbols – have tigliane skeleton and are found in croton oil from seeds of euphorbiacea ⇒ tumour promoters & anti-leukemic compound important use in cancer research ⇒ often esterified to C12 fatty acids esterified compounds are potent carcinogens can cause growth of cells to study cancer gibberellins (GA) – most studied of diterps with over 50 C20 & C19 GA known ⇒ ent-kaurene → important intermediate in GA synthesis “ent” seen as relative stereochemistry at positions 5 & 10 on the compound (A & B ring fusion point) but they are enantiomers Both found among diterps but ent- form used to make Gas ⇒ biosynthesis of gibberellins can be divided into 7 steps MVA → GGPP (committal step) Cyclization → ent-kaurene Oxidation of ent-kaurene Contraction of ring B → GA C20 → C19 GA Interconversions of GA Conjugation of GA ⇒ GA3 → most common gibberellin ⇒ usage includes: Promotes flowering Stem growth Breaking dormancy Enzyme synthesis Fruit development ⇒ commercial usages include: Inducing malting in barley - partially germinated barley; hydrolysis of starch to sugar → used in brewing to sustain fermentation Increase sugar cane yield – increases stock growth; 60 g/acre increases yield by 0.5 ton/acre (~10%) SESTERTERPENOIDS Sesterterps – C25 compounds made up of 5 isoprene units ⇒ found as linear, mono, bi-, tri-, tetra-, & macro-cyclic ⇒ rare compound & note very researched ⇒ important in host-microbe interactions In thaliana – lack of two root specific sesterterps had major impact on root microbiota assembly Leucosesterterpene – potential anti-angiogenic agent (anti-cancer) ⇒ geranylfarnesyl diphosphate (GFPP) – precursor for sesterterps (with additional isoprene unit) ⇒ made in plastids by GFPPS formed by plastid localized sesterterps synthases (SrTS) TRITERPENOIDS Triterpenoid – C30 compound made up of 6 isoprene unit ⇒ may be cyclic or acyclic Most are cyclic Usually tetra- or penta-cyclic Few tricycles No mono- or bi-cyclics ⇒ properties include Usually colorless High melting point Metabolically stable ⇒ most if not all triterps are derived from acyclic C30 squalene ⇒ can be divided into 4 groups True triterpenoids (include 4 & 5 ring compounds) Steroids Saponins Cardiac glycosides FPP + FPP undergo “head-to-head” addition to form squalene ⇒ FPP + FPP → squalene-2,3-epoxide = folded squalene epoxide → cyclic triterps & steroids Sterol skeleton synthesis ⇒ squalene undergoes modification by cyclase to fold to precyclization form (skeleton) ⇒ cyclase then cyclizes the compound to form sterol skeleton Cholesterol Pentacyclic – very common triterp found in waxy coatings of leaves, fruit & resins ⇒ also found in petroleum & waste products of oil refining ⇒ examples of pentacyclic triterps: β-amyrin (R=Me) vs Oleanic acid (R=COOH) → only difference is their R groups (C30) Limonoids – C30 with bitter tastes (in citrus) Hopanes – C30 compounds from cyanobacteria & plants; carries over to petroleum (marker in waste from refining & spills) Tetracyclic – consist of steroids & sterols with ring system of 3 C6 rings + 1 C5 ring ⇒ precursors to cholesterol Lanosterol (C30) – precursor in animals Cycloartenol (C30) – precursor in plants ⇒ same compounds except for cyclopropane (C9) Instead of double bond hydroxy at C3, maintained in most steroids there is a 8C chain at C17 ⇒ insects do not make steroids Get tetracyclic from diet followed by modification i.e., flamingo turning pink squalene – necessary in making steroids & sterols ⇒ folded squalene epoxide forms into cholesterol precursors (plant or animal) ⇒ carbons are removed and oxygen is added to form cholesterol (C27) ⇒ cholesterol can then form many compounds such as: vitamin D3 Bile acids (C24) Steroid hormones (C21, 19, 18) Insect hormones (C27) Plant steroids (C21, 19, 18) Sapogenins (C27) Alkaloids (C27) Saponins – large class of triterps ⇒ diosgenin – from wild yams (dioscorea sp.) ⇒ hecogenin – from Agave Sp. (C27 – sterol) Surface active agent Soap like – cause forming (i.e., root beer) Can hemolyze blood cell Used in fish poisons, anti-fungal & anti-biotics Usually in glycoside form ⇒ solanine – sterol alkaloid & glycoside Found in green parts of a potato Can cause nausea or paralysis → potentially fatal Cardiac glycosides – found in several plant species & is formed from pregnenolone + acetyl CoA ⇒ usually, glycoside → with unusual sugar attached at C3 Genin name given to aglycoside (without sugar) ⇒ active when sugar is removed Inhibits Na/K-pump in cell membrane ⇒ found in digitalis (foxglove) ⇒ examples include (all impact the heart): Cardenolides (C23) – digitoxigenin that forms into digitoxin with hydroxylation at C12 Bufadienolides (C24) – found in toad poisons and a few plant species; too toxic to use as cardiac treatments Ouabin (strophanthin G) – potent cardiac glycoside → a rhamnoside Oleandrin – found in oleander & deadly if ingested Convallatoxin – most toxic of all CG and can be found in lilly of the valley (deadly) TETRATERPENOIDS Carotenoid – C40 terpenoid (8 isoprene units) that is not made in animals ⇒ carotenoid & their precursors are widely distributed & important compounds ⇒ Biosynthesis is based on condensation of 2 units of GGPP Chain does not grow but modifications result in large changes to compound i.e., introducing double bonds ⇒ biosynthesis occurs in Chloroplast of plants & algae Chromoplasts (i.e., tomato & other fruit) Photosynthetic bacteria) ⇒ not synthesized in animals Intake by diet → accumulated & stored in animals i.e., flamingos, starfish, lobsters etc. lose color without dietary source of carotenoids ⇒ properties include Yellow, red, orange colour in leaves, fruits & flowers → 400-500nm absorption Lipid soluble (insoluble in water) Important commercial coloring agent ⇒ lycopene – normally found in multivitamins & plays large role in general health Antioxidant properties Xanthophyll-zeaxanthin cycle – response to light ⇒ violaxanthin – present in low light Loses oxygen to form intermediate ⇒ antheraxanthin – intermediate ⇒ zeaxanthin – present in high light Loses oxygen to form intermediate CYTOCHROME P450 & TERP DIVERISTY Plant P450 families are categorized into 11 clans ⇒ P450s involved in specialized terpenoid biosynthesis are spread all across 11 families Terpene alcohols & oxidized terpenoids formed by TPSs & P450s undergo diversification by addition of acyl, glycosy­­­­­l, benzoyl, or even alkaloid groups