Lecture 1-3.docx

QUIZ 1 REVIEW LECTURE 1 ENVIRONEMNTAL & NATURAL PRODUCTS Secondary metabolites not primary metabolites ⇒ 2° does not mean unimportant ⇒ produced along offshoots of primary pathways ⇒ often induced by varying environmental conditions Secondary metabolites may have significant ecological, econo...

QUIZ 1 REVIEW LECTURE 1 ENVIRONEMNTAL & NATURAL PRODUCTS Secondary metabolites not primary metabolites ⇒ 2° does not mean unimportant ⇒ produced along offshoots of primary pathways ⇒ often induced by varying environmental conditions Secondary metabolites may have significant ecological, economic, & pharmacological uses There is no dividing line between 1° and 2° metabolism ⇒ both processes are closely connected Primary metabolism provides chemical building block for all important 2° metabolites 3 major biosynthetic pathways act as starting points for many 2° metabolites ⇒ Shikimic Acid Pathway i.e., lead to amino acids & aromatics ⇒ Mevalonate (isoprenoid) Pathway i.e., terpenes, steroids, & carotenoids ⇒ Acetate-Malonate Pathway i.e., polyketides (leads to polyphenolics), & fatty acids CRITERIA TO DISTINGUISH SECONDARY METABOLITES Restricted distribution mainly in plants & microorganisms ⇒ generally characteristic of particular families, genera, or species Formed along special biosynthetic pathways Usually considered to be marginal or non-essential to primary life processes BIOLOGICAL FUNCTIONS OF 2° METABOLITES Attract insect & bird pollinators to flowers Repel predators by smell, taste, toxicity Act as phytoalexins (antibiotics) in plants Reduced competition from other plant species ⇒ i.e., allelopathy Formed to detoxify potentially harmful compounds Storage products that can be catabolized for energy for simpler metabolites EXAMPLES OF NATURAL PRODUCTS USED IN MEDICINE<M AGRICULTURE< & INDUSTRY Vitamin E (α-tocopherol), β-carotene (precursor of Vitamin A) & ergosterol (precursor of Vitamin D) ⇒ all products of the isoprenoid pathway High dosages of Vitamin E & β-carotene are recommended for antioxidant values (effective scavengers of ROS) Insecticides ⇒ nicotine → alkaloid) ⇒ rotenone → isoprenoid pathway ⇒ pyrethrin → polyketide, isoprenoid pathway ⇒Spinosad & Avermectins → polyketide pathway Waxes & oils ⇒ for cosmetics & paints → variety of sources Dyes – for food & cloth ⇒ red, orange, blue, purple, yellow, & brown → from many sources Spices & flavours ⇒ e.g., Vanilla, peppermint, orange, lemon, clove, cinnamon Perfumes ⇒ Largely isoprenoids Pharmaceuticals ⇒ cascara sagrada & senna extracts → anthraquinones, laxatives ⇒ castor oil → polyketide, laxative ⇒ steroids → isoprenoids ⇒ codeine → alkaloid, analgesic ⇒ morphine → alkaloid, narcotic ⇒ Digitoxin → cardiac glycoside (heart disease) ⇒ Ephedrine → alkaloid, bronchodilator ⇒ Taxol → mixed synthesis, anticancer drug MANY PLANT PRODUCTS ARE POISONOUS Many familiar foods & drinks may contain substances that can cause illness or may be carcinogen ⇒ chamomile tea → can cause anaphylactic shock or allergic rhinitis in sensitive individuals ⇒ apples → may contain relatively high amounts of formaldehyde ⇒ broccolis → contain a compound not unlike dioxin ⇒ peanuts → can be contaminated by carcinogenic fungus ⇒ potato peels → may contain high levels of solanine which block cholinesterase i.e., just as the organophosphate insecticides Many animals (including humans) have developed metabolic defenses → protect us against moderate doses of substances ⇒ toxins that plant produce as protection → may be present in amounts lethal to humans i.e., environmental stress or attacks by organism can trigger large increase in natural pesticide production Many fruit & veggies also produce compound that are natural cancer inhibitors ⇒ visualize a balance in our natural food supply between cancer-causing & cancer inhibiting substances Must be cautious in efforts to bio-engineer disease-resistant & insect-resistant varieties ⇒ potato variety developed with high resistance to insect attack had to be withdrawn as it was too toxic for human consumption REVIEW OF CHEMICAL ISOMERISM Different molecules with the same chemical formula Isomerism is very important in natural product & environmental biochemistry ⇒ many natural compounds have complex structures with many chiral centers ⇒ e.g., 46% C2H6O CH3CH2OH CH3OCH3 ⇒ enzymes add groups stereochemically Steroid hydroxylation ⇒ enzymes only recognize one form of a structural isomer or stereoisomer E.g., only L-amino acid used in biology Aspartame (D-Asp-L-Phe) → sweet flavour, not recognized by enzyme (no calories) STRUCTURAL ISOMERS Same molecular formula with different arrangement of atoms & different physical properties ⇒ not just mirror images Alkanes, alkenes, & alkynes <4C do not have isomers ⇒ CH4, CH3CH3 & CH3CH2CH3 Functional groups (=O, OH, Cl, etc.) can alter this requirement Must be different configuration not just a different conformation ⇒ atoms arranged differently in space with respect to each other STEREOISOMERS Same bond order, different arrangement of atoms in space ⇒ same chemical formula if spatial arrangement was not indicated ⇒ CHCl=CHCl could be geometric or optical isomers Optical – same physical properties, except rotation of polarized light Geometric – different physical properties OPTICAL ISOMERS Have one or more chiral centers → asymmetric carbons Enantiomers – stereoisomers that are not superimposable (NSMI) ⇒ have identical properties but rotate polarized light in opposite directions Melting/boiling points, density, absorption spectrum, etc. ⇒ same amount of rotation is opposite directions ⇒ equal amounts of enantiomers in a mixture Racemic mixture Optically inactive (net rotation = 0) ⇒ identical chemical reactivities except in present of another stereoisomer (i.e., enzymes) Optically active – rotate plane polarized light ENZYME CLASSIFICATION Enzymes do not cause new type of organic reactions ⇒ speed up reactions that are thermodynamically & mechanistically possible Enzymes control reactions in 2° just as they do in 1° Brief look at pathways & literatures reveals enzymes often not identified (thousands) ASSUMPTIONS ABOUT ENZYMES Enzymes are proteins They do not allow new kinds of organic reactions “Black Boxes” that speed up allowed organic reactions and let them occur with great specificity ⇒ lower kinetic barriers of reaction ⇒ speed up to 1012-fold ⇒ do not alter equilibrium or ∆G of reactions Enzymes can only catalyze a specific rxn Enzyme is selective to a specific substrate or small group of substrates Enzyme will only produce one product or a small group of products Each enzyme only accepts one enantiomer and produces a stereo specific product 3D structure of enzyme & co-factors makes reaction state intermediate possible Many cases, acid/base catalysis is key to enzyme function using acid and/or basic AA COMMON CO-SUBSTRATES ATP – energy source CoASH – activated group carrier NAD(P)H – reducing power ⇒ i.e., use of ATP to drive a reaction (like ligase reaction) AMP – good leaving for nucleophilic attack on Y ENZYME CLASSES Oxidoreductases – catalyze redox reactions by transferring electron from donor to receptor (reduced) ⇒ oxidation reduction reactions (redox) Oxidation → loss of electrons from a chemical Reduction → gain of electrons by a chemical ⇒ dehydigenases – H added or removed but O2 not involved (loss of H) ⇒ oxidases – O2 serves as the electron acceptor & either H2O or H2O2 is forms (H to O acceptor) Polyphenol oxidases – causes browning of certain fruits/veggies Peroxidases – oxidases use H2O2(O) as e-acceptor Catalase – oxidases that use H2O2(O) as donor & acceptor ⇒ polyphenol oxidase – cause browning of cut apples, potatoes, avocados (in fungi & higher plants) Oxygen not added to substrate, electron transferred to O2 Reaction inhibited by low pH because first step is base catalyzed ⇒ peroxidases – oxidases that use H2O2 (O) as electron acceptor ⇒ catalases – oxidases that use H2O2 (O) as donor & acceptor ⇒ oxygenase’s – oxygen incorporated into substrate as oxidation occurs (o added) Monooxygenases → mixed function where one O atom is in product other in H2O (e.g., most are cytochrome P450) Dioxygenases & lipoxygenases – from plants & are good examples of O2 incorporation (catalyzes addition of O2 to form hydroperoxides ⇒ reductases – generally the reverse of dehydrogenase where an apparent dehydrogenase reaction is pushed to the left (o removed or H added) If principal component in reaction is reduced (NAD(P)H is oxidized usually) → enzyme is called a reductase Transferases – transfer groups containing C, N, P, or S from one substrate to another (no redox) ⇒ kinases – transfer of Pi from ATP to an acceptor; two types recognized Transfer among high energy compounds – creatine phosphate is a high energy carrier & energy storage source; ATP can now be used for energy in cell ⇒ phosphorylases – transfer glycosyl residues from starch (plant + animal) to Pi Starch + nPI → (starch & phosphorylase) → n G-1-P ATP is not involved but energy in glycosidic bone Is preserved ⇒ aminotransferases – transamination reactions (i.e., transfer -NH2) specified by 2 amino acids involved or by the donor Glutamate is most common donor of NH2 groups ⇒ methyltransferases – transfer CH3 groups S-adenonsyl-methionine (SAM) is primary source of CH3 groups in methylation reactions ⇒ acetyltransferases – transfer acetyl group from acetyl CoA Hydrolases – catalyzes hydrolysis of CO & CN bonds ⇒ glycosidases – break glycosidic bonds (fundamental bond in polysaccharides & most di- & oligo-saccharides Invertase → splits sucrose β-glucosidase → splits sugar from cyanogenic glycoside β-galactosidase → splits sugar from indole to form blue coloured compound ⇒ phosphatases – hydrolyze esters of phosphoric acid & made up of 3 subgroups (Alkaline, neutral, & acid phosphatases) Mono-phosphatases (esterase’s) → hydrolyze Mono-phosphoric esters (e.g., Calvin cycle) Di-phosphatases (phosphodiesterase’s) → include DNAses, cyclic AMP phosphor-diesterase, & snake venom diesterases, activity also in self-splicing RNA ⇒ lipases – hydrolyze triglycerides (fats) to glycerol & free fatty acids ⇒ amylases – hydrolyze starch in storage cells to maltose units α-amylase → cleaves α 1→4 bonds of amylose & amylopectin to yield maltose & limit dextrin β-amylase → removes maltose units at non-reducing end of the glucan; degrades amylose completely but amylopectin to 1→6 bonds Isoamylase → cleaves amylopectin at 1→6 ⇒ protease – cleaves peptide bonds peptidase → hydrolyze peptides at terminal amino acids (exopeptidases) both N & C Proteinases → cleave at internal peptide bonds (endopeptidases) Lyases – non-hydrolytic removal of group (e.g., NH2) often forming a double bond or addition of group across ⇒ decarboxylase ⇒ aldolase ⇒ lyase (deaminase) – i.e., deamination by PAL ⇒ fumarase – hydrases & hydratases Isomerases – transform substrate into an isomer ⇒ no net changes in redox or chemical functionality ⇒ includes racemases, epimerases, mutases Ligases (synthetases) – condensing enzyme catalyze union of two molecules using ATP or another nucleoside triphosphate ⇒ carboxylase – with carboxylation of pyruvate as an example Provides OAA in TCA cycle ⇒ synthetase – with formation of an amide by incorporation of NH4+ into an amino acid Acetyl-CoA synthetase (acy-x-synthetase) → catalyze a major reaction for primary & secondary metabolism where ATP drives formation of thioester bond LECTURE 2 TERPENOIDS Terpenoids (isoprenoids) – very large, diverse groups of compounds with all common origin ⇒ may contain 5-C to several hundred carbons arranged in cyclic & acyclic compounds ex. essential oils, steroids, rubber, many flavours, & medicinal compounds ⇒ term refers to all compounds built of isoprene units regardless of additional or missing carbons or functional groups terpenes – refer specifically to hydrocarbon compounds composed of basic 5-C isoprene unit or multiple isoprene units Isoprene rule – states all terpenes are multiples of 5-C isoprene ⇒ Biogenic isoprene rule – states all terpenoids are synthesized from a common 5-C precursor Final structures cam result from chemically reasonable modifications of an integral number of the 5-C precursor units Isopentyl Pyrophosphate (IPP) – known as active isoprene, is an actual biosynthetic precursor of terpenoids ⇒ formed from mevlonate Terpenoids grouped according to number of 5-C units required for their synthesis ⇒ hemiterpenoids – 1 isoprene unit (C5) ⇒ monoterpenoids – 2 isoprene units (C10) ⇒ sesquiterpenoids – 3 isoperen units (C15) ⇒ diterpenoid – 4 isoprene units (C20) ⇒ triterpenoid – 6 isoprene units (C30) ⇒ tetraterpenoids – 8 isoprene units (C40) ⇒ polyterpenoids – “n” isoprene units (Cn*5) Synthesis of some terpenoids occurs in all organisms ⇒ synthesis by acetate-mevalonate pathway ubiquitous to all organisms ⇒ only angiosperms can elaborate all types however Ex. carotenoids are not made by animals & sterols are not made by insects in general terpenoids are ⇒ lipid soluble ⇒ located in the cytoplasm & not the vacuole ⇒ extracted with methanol, petroleum ether, diethyl ether, chloroform i.e., polar to moderately polar organic solvents BIOSYNTHESIS OF C5 UNIT mevalonic acid (MVA, mevalonate) – primary precursor of IPP & therefore all terpenoids ⇒ derived from condensation of three molecules of acetyl-CoA mva molecule → only use 3R enantiomer in biosynthesis ⇒ presence of prochiral centers allowed chemists to establish that each step of subsequent synthesis of C5 & C10 isoprenoids is stereospecific prochiral carbon – achiral carbon that became chiral when one of its ligands changed ⇒ in the case of C5 in MVA, HR is replaced by deuterium or tritium and C5 will be chiral with R configuration therefore Hr & HS are called pro-R or pro-S hydrogens (ligands) of C5 pro-ligands are most commonly (but not always) H atoms biosynthesis of MVA (subsequently IPP) begins with condensation of two molecules of acetyl-CoA via action of transferase ⇒ 3rd acetate added via synthase ⇒ note: different pathway in chloroplast MEP (methyl erythritol phosphate) pathway – from GA-3-P & pyruvate to form IPP notes on biosynthesis of MVA pathway ⇒ Step 1 is a transfer of acetyl groups from one acetyl-CoA to another ⇒ Step 2 is a condensation reaction (via lyase) with subsequent cleavage of CoA (same mechanism as oxaloacetate to citrate in CAC) ⇒ reduction of HMG-CoA to MVA is essentially irreversible in vivo 4e- reduction with 2 NADPH ⇒ HMG-CoA reductase is probably the principle point of regulation in isoprenoid biosynthesis ⇒ reductase is bound to endoplasmic reticulum & apparently regulated in 3 ways: Inactivated via phosphorylation by kinase (transferase) & activated by phosphates (hydrolase) By feedback control of mRNA for enzyme Enzyme turnover (synthesis & degradation) regulated by feedback control ⇒ competitive inhibition of HMG-reductase by lovastatin is common medical treatment for controlling high levels of cholesterol in humans ⇒ lovastatin is a natural product obtained from fungi ⇒ MVA only used to form terpenoids (no other significant fate) ⇒ Step 4 & 5 is where MVA is phosphorylated using 2 ATPs via action of MVA kinase (another point of regulation) ⇒ Step 6 is a 2-part reaction using 3rd molecule of ATP to phosphorylate OH group ⇒ Pi is a good leaving group ⇒ CO2 is also lost to yield IPP (one of two types of active isoprene) ⇒ equilibrium of IPP:DMAPP is approximately 3;97 ⇒ migration of double bond in isomer (3,2 shift) occurs via electrophilic attack by H+ on methylene carbon in IPP Carbon cation produced forces a shift in double bond with loss of H+ from C2 IPP & DMAPP are biosynthetic functional equivalents of isoprene ⇒ two key hemiterpenoids ⇒ their union leads to subsequent production of all higher molecular weight terpenoids DMAPP – excellent alkylating agent (effective electrophile) ⇒ electron “pressure” in allylic group of DMAPP facilitates loss of PPi Resulting positive charge is dispersed over allyl radical (carbon cation is resonance stabilized) ⇒ under influence of DMA transferase → C1 at tail of DMAPP undergoes nucleophilic attack by pi-electrons in head of IPP Tail to head condensation yields C10 → GPP (trans-geranyl-PP) Shift in double bond of IPP to yield DMAPP configuration at tail of GPP ⇒ chain can be lengthened by nucleophilic attack of another IPP on GPP to yield C15 FPP Repetition of tail to head condensation with different enzymes lead to polymers ⇒ i.e., rubber (mixture 1,4-polyisoprene) ⇒ Many plants produce latex from which rubber is obtained but commercial source is Hevea Brasiliense Rubber shows a wide spectrum of molecular weights → consisting of 1500 to 60,000 isoprene (prenyl) residues Tail to head condensation also holds for mono (C10), sesqui (C15) & diterpenes (C20) ⇒ in contrast, mechanism of formation for the tri- & tetra- terpenoid involves tail to tail condensation BIOSYNTHETIC RELATIONSHIPS OF TERPENOIDS HEMITERPENOIDS Found in.. ⇒ all organisms ⇒ in extracts (following loss of PPi) ⇒ in plant oils Especially evergreens Detectable in forest air Functions include ⇒ DNA crosslinking ⇒ can be taken orally + sunlight promotes tanning ⇒ treatment for psoriasis ⇒ used in blood sterilization LECTURE 3 MONOTERPENOIDS Monoterpenoids – made up of 2 isoprene units (C10) ⇒ source of most monoterpenoids → C10 Geranyl-PP (GPP) ⇒ conveniently divided into Acyclic Monocyclic Bicyclic Iridoids Irregular & mixed ⇒ larger number of compounds in plants (>100) ⇒ almost every possible arrangement of 2 isoprene’s ⇒ widely distributed ⇒ some found in insects, rare in other organisms Major constituents of essential oils ⇒ significant economic importance → flavours, perfumes, solvents Functions in plants generally ecological (not physiological) ⇒ competition with other plants (herbicides) ⇒ insect attractants & repellants Attraction → pollination Repelling → pesticides Characteristics: ⇒ colorless (usually) ⇒ lipid soluble ⇒ volatile & fragrant ⇒ steam distillable (gas chromatography) ⇒ readily separated and analyzed with GC ACYCLICS Monoterpenoids that do not form complete cyclic structure ⇒ formed from IPP + DMAPP Nerol – in orange blossom Geraniol – in citronella oil & rosemary oil ⇒ can be toxic Citral – mixture of geranial & neral ⇒ found in many plants (e.g., lime) ⇒ uses include Citrus smell Can be used in baking (food flavor) Can be used as coloring Aldehyde – strong lemon ⇒ in rose oil & lemon grass oil GPP undergoes ionization → α-terpinyl cation ⇒ forms many compounds including bi-cyclics MONOCYCLICS Most cyclic derived from → neryl-pp (neryldiphosphate) ⇒ folded such that cyclization favored ⇒ final products form modification & introduction of functional groups P-menthane skeleton common to most monocyclics ⇒ core skeleton undergoes cyclization to form many compounds Piperitenone + 2H → Pulegone + 2H → Menthone + 2H → Menthol Menthol makes up 70% of peppermint oil Limonene – colorless, important mono-cyclic p-menthane ⇒ rearrangement of double bond compared to NPP ⇒ often exists as a racemic mixture ⇒ found in citrus Key constituent of citrus oil Gives lemon flavour ⇒ also found in turpentine, caraway, & dill ⇒ example of where nature uses same compound in several instances Male boll weevil (pest) attracted to cotton by limonene Thymol: major aromatic monoterpenoid ⇒ a phenol (2-isopropyl-phenol) ⇒ essential oil from thymus vulgaris Pungent caustic taste of thyme ⇒ mildly toxic to animals Rat LD50 is 1g/kg ⇒ good fungicide Perhaps why plants make it ⇒ used as preservative BICYCLICS Large group divided into 7 classes First class derived from limonene ⇒ α-pinene (more favourable structure) & β-pinene (less favourable) Similar compounds with different effects ⇒ chief constituents of commercial terpentine Limonene & sesquiterpenoids also present in oils from many pines species ⇒ amounts vary with pine species ⇒ age influences quantity Varies from tree to tree Varies with age & environmental conditions Camphor – “mothballs” that come from camphor trees or commercially manufactured from pinere ⇒ good plasticizer ⇒ used in Original celluloids Explosives, varnishes, & antiseptics Cineole – chief constituent of eucalyptus oils ⇒ used in Antiseptics Treatment for respiratory disease In halls (cough drops) → mentholyptus Ascaridole – found in the plant Chenopodium; weed that is now cultivated ⇒ derived from endo-peroxidation of limonene ⇒ used as Laxative → expels intestinal worms Insecticide for the plant ⇒ has Parasitical effects Drug resistance MECHANISM OF α-PINENE & β-PINENE BIOSYNTHESIS Forms carbocation intermediate ⇒ carbon atom on NPP becomes positively charged when OPP- leaves Nature of carbocation is unclear ⇒ short lived ⇒ stabilized by enzyme ⇒ transition states intermediates (not proven) ⇒ partly because enzymes in low concentration INTRAMOLECULAR REARRANGEMENTS Variety of compounds can be made ⇒ electron transfer that can happen to support variety of compounds being made What gives variety of compounds? ⇒ carbocation intermediates ⇒ intermolecular rearrangement IRIDOIDS Iridoids – also known as monoterpene lactones; usually have 6 membered lactone ring fused to 5 membered ring ⇒ derived from NPP but biosynthetic pathway not fully worked out (synthesized in labs) Organic synthesis has been performed ⇒ occurs as β-D-glucoside → water soluble ⇒ loganin – C11 compound due to methyl-ester Becomes catnip if glucose, OH, & MeOH removed Progenitor of iridate family ⇒ uses include: Repellant & irritant (chemical defence for insects) ⇒ precursor to monoterpenoid alkaloids (indoles) Natural insecticides ⇒ found in many plants Distributed throughout the plant (not organ specific) Secoiridoids – cyclopentane ring of iridoid replaced by second 6 membered lactone ring ⇒ cyclopentane ring cleaved to give the extra lactone ring ⇒ has similar properties to iridoid IRREGULAR & MIXED Members of this group have restricted distribution ⇒ biogenic anomalies ⇒ most are C10 but unusual pathways Camphene – looks like a bicyclic monoterpene but not formed from NPP ⇒ formed from Lavandula skeleton → comes from linkage of 2 DMAPPs In normal cases would be formed from IPP + DMAPP In this case departure of IPP leads to irregularity Pyrethrin – example of mixed monoterpenes derived from chrysanthemum flowers (chrysanthemyl skeleton) ⇒ R group is a ring group with polar constituent ⇒ cyclopropane ring is strained & readily undergoing opening ⇒ non-toxic to plant & animals Less sensitive sodium channels in their nervous systems ⇒ toxic to fish and insects Insects have more sensitive nervous system sodium channels Synthetic analogues used as insecticides (environmentally safe)