BIOL 158: BIOLOGICAL CHEMISTRY II

Transcription

1 BIOL 158: BIOLOGICAL CHEMISTRY II Lecture 9: TERPENES Lecturer: Christopher Larbie, PhD

2 The volatile component of fragrant materials in plant belong to a class of compounds called terpenes. Terpenes/terpenoid compounds are widely distributed in nature mostly in plants. They are polymers of isoprene units joined head to tail. An isoprene unit contains 5-carbon atom put together in a regular pattern, usually head-to-tail in terpenes up to 25 carbons The terpenes have been long associated with the term Essential Oils comprising resins, steroids and rubber

3 In fact, they are hydrocarbons that usually contain one or more C=C double bonds, while the terpenoids are oxygen-containing analogues of the terpenes They are thoroughly distributed in the plant kingdom, especially in those plants that have abundant chlorophyll 2-Methyl-1,3-butadiene

4 The double bond in the isoprene units are in the trans geometric configuration. Isoprene units are produced from acetyl CoA or acetic acid Terpenes can be cyclic or open chain molecules, carboncarbon saturated or unsaturated and may contain a variety of functional groups such as aldehydes, alcohols, hetons and esters Terpenes occur in rubber, carotenoids in carrots, potatoes, yellow vegetables, vitamin A, E and K, chlorophyll and urbiquinone.

5 Terpenes containing 30 carbons or more are usually formed by the fusion of two smaller terpene precursors such that the head-to-tail "rule" appears to be violated In overall, terpenes hold potential interest practical applications especially in the fragrance and flavour industries, as well as in the pharmaceutical and chemical industries.

6 Classification of Terpenes No of C No of Isoprene units Class of terpenes 5 1 Hemeterpene 10 2 monoterpenes 15 3 sesquiterpenes 20 4 diterpenes 25 5 sesterterpenes 30 6 triterpenes eg. Steroids 40 8 tetraterpenes eg. Carotenoids polyterpenes eg. Rubber

7 Methods of extraction 1. Expression; squeezing the juice out of plant material 2. Steam distillation; suitable for the lower members especially up to 20 carbon compounds. 3. Extraction with volatile solvent; vacuum distillation and chromatographic method.

8 Isoprene Rule A compound is said to be a terpenoid if it can be broken into 5-carbon units usually joined head to tail Example

9 Examples of Terpenes Monoterpenes are naturally occurring compounds, the majority being unsaturated hydrocarbons (C10) But, some of their oxygenated derivatives such as alcohols, ketones, and carboxylic acids are known as monoterpenoids The branched-chain C10 hydrocarbons comprises of two isoprene units and is widely distributed in nature with more than 400 naturally occurring monoterpenes identified

10 Moreover, besides being linear derivatives (Geraniol, Citronellol), the monoterpenes can be cyclic molecules (Menthol monocyclic; Camphor bicyclic; Pinenes (α and β) Pine genera) as well Thujone (a monoterpene) is the toxic agent found in Artemisia absinthium (wormwood) from which the liqueur, absinthe, is made Borneol and camphor are two common monoterpenes. Borneol, derived from pine oil, is used as a disinfectant and deodorant Camphor is used as a counterirritant, anaesthetic, expectorant, and antipruritic, among many other uses

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12 Sesquiterpenes Sesquiterpenes are biogenetically derived from farensyl pyrophosphate and in structure may be linear, monocyclic or bicyclic They constitute a very large group of secondary metabolites, some having been shown to be stress compounds formed as a result of disease or injury One group is the Sesquiterpene Lactones. Over 500 compounds of this group are known; they are particularly characteristics of the Compositae but do occur sporadically in other families

13 Not only have they proved to be of interest from chemical and chemotaxonomic viewpoints, but also possess many antitumor, anti-leukaemic, cytotoxic and antimicrobial activities They can be responsible for skin allergies in humans and they can also act as insect feeding deterrents. Hemigossypol and related aldehydes together with the dimeric gossypol are sesquiterpenes stress compounds found in the subepidermal glands, immature flower buds and seed kernels of the cotton plant (Gossypium spp.) Besides having insecticidal properties, gossypol is of considerable pharmaceutical interest in that in humans, it functions as a male anti-infertility agent

14 In China, gossypol has been used to treat infertility in males and studies have shown an increase in sperm production

15 Diterpenes: The diterpenes, which contain 20 carbons (four C5 units), include phytol (the hydrophobic side chain of chlorophyll), the gibberellin hormones, the resin acids of conifer and legume species, phytoalexins, and a host of pharmacologically important metabolites, including taxol, an anticancer agent found at very low concentrations (0.01% dry weight) in yew bark, and forskolin, a compound used to treat glaucoma Some gibberellins have only 19 carbon atoms and are considered norditerpenoids since they have lost 1 carbon through a metabolic cleavage reaction Vitamin A, which combines with protein opsin to form the active pigment of the retina

16 Triterpenes These contain 30 carbon atoms, are generated by the head-to head joining of two C15 chains, each of which constitutes three isoprene units joined head-to-tail This large class of molecules includes the brassinosteroids, the phytosterol membrane components, certain phytoalexins, various toxins and feeding deterrents, and components of surface waxes, such as oleanolic acid of grapes Squalene in shark liver oil, smaller quantities are in olive oil, rice bran oil, yeast, wheat oil. Squalene is used in biosynthesis of steroids

17 CH 3 CH 3 CH 3 H 3 C CH 3 H 3 C C H 3 C H 3 Squalene (acyclic) H HO -amyrin in resin (cyclic)

18 Tetraterpenes: The most prevalent tetraterpenes (40 carbons, eight isoprene units) are the carotenoid accessory pigments which perform essential functions in photosynthesis. Polyterpenes: These contain more than eight isoprene units, include the prenylated quinone electron carriers (plastoquinone and ubiquinone), long-chain polyprenols involved in sugar transfer reactions (e.g., dolichol), and enormously long polymers such as rubber (average molecular mass greater than 10 6 Da), often found in latex Natural rubber may be regarded as l, 4 adolition polymer, it is treated with elemental sulphur in a process called vulcanization

19 Biosynthesis of Terpenoid compounds The biosynthesis of all terpenoids can be divided into 4 steps 1. Synthesis of the fundamental precursor, Isopentenyl pyrophosphate (IPP) 2. Repetitive addition of IPP to form a series of prenylpyrophosphate homologs, which serve as the immediate precursors of the different classes of terpenoids.

20 3. Elevation of these allylic prenylpyrophosphates by specific terpenoid synthases to yield terpenoid skeletons. 4. Secondary modification of the skeletons (largely redox reactions) to give rise to the functional properties and great chemical diversity of this family of natural products

21 Compartmentation A more fundamental, and perhaps universal, feature of the organization of terpenoid metabolism exists at the subcellular level. 1. The sesquiterpenes (C15), triterpenes (C30), and polyterpenes appear to be produced in the cytosolic and endoplasmic reticulum (ER) compartments. 2. Isoprene, the monoterpenes (C10), diterpenes (C20), tetraterpenes (C40), and certain prenylated quinones originate largely, if not exclusively, in the plastids

22 The evidence now indicates that the biosynthetic pathways for the formation of the fundamental precursor IPP differ markedly in these compartments, with the classical acetate/mevalonate pathway being active in the cytosol and ER and the glyceraldehyde phosphate/pyruvate pathway operating in the plastids

23 The acetate/mevalonate pathway 1. Hydroxymethylglutaryl-coenzyme A (HMG-CoA) is the precursor for terpenoid synthesis HMG-CoA is also an intermediate on the pathway for synthesis of ketone bodies from acetyl-coa. The enzymes for ketone body production are located in the mitochondrial matrix HMG-CoA destined for terpenoid synthesis is made by equivalent, but different, enzymes in the cytosol. HMG- CoA is formed by condensation of acetyl-coa and acetoacetyl-coa, catalyzed by HMG-CoA Synthase

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25 2. HMG-CoA reductase catalyzes production of mevalonate from HMG-CoA The carboxyl group of hydroxymethylglutarate that is in ester linkage to the thiol of coenzyme A is reduced first to an aldehyde and then to an alcohol NADPH serves as reductant in the 2-step reaction. Mevaldehyde is thought to be an active site intermediate, following the first reduction and release of CoA. HMG- CoA reductase is an integral protein of endoplasmic reticulum membranes The catalytic domain of this enzyme remains active following cleavage from the transmembrane portion of the enzyme

26 3. Mevalonate is phosphorylated by 2 sequential phosphate transfers from ATP, yielding the pyrophosphate derivative

27 Glyceraldehyde phosphate/pyruvate pathway In plastids, pyruvate reacts with TPP to yiel a 2-C fragment, hydroxylmethyl-tpp which condenses with glyceraldehyde 3-phosphate

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29 Synthesis of Terpenoids 1. Pyrophosphomevolanate decarboxylase catalyzes ATP-dependent decarboxylation, with dehydration, to yield isopentenyl pyrophosphate Isopentenyl pyrophosphate isomerase inter-converts isopentenyl pyrophosphate and dimethylallyl pyrophosphate The mechanism involves protonation followed by deprotonation

30 2. Prenyl transferase catalyzes a series of head-to-tail condensation reactions Dimethylallyl pyrophosphate reacts with isopentenyl pyrophosphate to form geranyl pyrophosphate Condensation with another isopentenyl pyrophosphate yields farnesyl pyrophosphate. Each condensation reaction is thought to involve a reactive carbocation formed as PPi is eliminated Prenyl transferase (Farnesyl Pyrophosphate Synthase) has been crystallized with the substrate geranyl pyrophosphate bound at the active site

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32 2. Squalene synthase catalyzes head-to-head condensation of 2 molecules of farnesyl pyrophosphate, with reduction by NADPH, to yields qualene

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34 Tracer studies using 14 C have confirmed that terpenoid compounds are all formed initially through acetate mevalonate pathway It has been shown that steroids are modified terponoid compound of cholesterol C27 may be regarded as C30 C3. Carotenoids which are C40 may be regarded as terpenoid compounds Role of IPP To prevent esterification of the coo-funcctional group and makes the ester a good leaving group.

35 Carotenes The term carotene (also carotin, from the Latin carota, or carrot) is used for several related unsaturated hydrocarbon substances having the formula C 40 H x, which are synthesized by plants but cannot be made by animals Carotene is an orange photosynthetic pigment important for photosynthesis Carotenes are all coloured to the human eye. They are responsible for the orange colour of the carrot, for which this class of chemicals is named, and for the colours of many other fruits and vegetables (for example, sweet potatoes, chanterelle and orange cantaloupe melon)

36 Carotenes are also responsible for the orange (but not all of the yellow) colours in dry foliage They also (in lower concentrations) impart the yellow coloration to milk-fat and butter Omnivorous animal species which are relatively poor converters of coloured dietary carotenoids to colourless retinoids have yellowed-coloured body fat, as a result of the carotenoid retention from the vegetable portion of their diet The typical yellow-coloured fat of humans and chickens is a result of fat storage of carotenes from their diets

37 Carotenes contribute to photosynthesis by transmitting the light energy they absorb to chlorophyll They also protect plant tissues by helping to absorb the energy from singlet oxygen, an excited form of the oxygen molecule O 2 which is formed during photosynthesis β-carotene is composed of two retinyl groups, and is broken down in the mucosa of the human small intestine by β-carotene 15,15'-monooxygenase to retinal, a form of vitamin A β-carotene can be stored in the liver and body fat and converted to retinal as needed, thus making it a form of vitamin A for humans and some other mammals

38 The carotenes α-carotene and γ-carotene, due to their single retinyl group (β-ionone ring), also have some vitamin A activity (though less than β-carotene), as does the xanthophyll carotenoid β-cryptoxanthin. All other carotenoids, including lycopene, have no betaring and thus no vitamin A activity (although they may have antioxidant activity and thus biological activity in other ways)

39 α-carotene γ-carotene

40 All carotenes absorb in the UV/VIS region; it means that UV can be used to quantitate carotenes in natural compounds

41 Retinol Carotenoids are tetraterpenes, widely distributed in both plants and animals. They are called lipodromic pigment meaning fat soluble They are coloured, yellow violet orange or red. In higher plants, carotenes are found in the leaves and chlorophyll They constitute the principal pigments of certain yellows, orange and red flowers Types of Carotenoids Carotenes - hydrocarbons soluble in pet ether. Xanthophylls - oxygenated derivatives of the hydrocarbons of

42 STEROIDS Steroids are compounds when distilled in the presence of selenium give a hydrocarbon called cyclopentano perhydro phenanthrene ring Classification of Steroids Sterols Bile acids Sex hormones Adrenal cortex hormones Steroidal glycosides Hydrocarbons

43 Sterols These are steroids with alcoholic OH functional group. Sterols are widely distributed in nature They may occur as free compounds or as esters of some fatty acids e.g. cholesterol most abundant of sterols. Cholesterol in the precursor of all steroids in animals. Cholesterol is a white crystalline solid

44 Features of sterols 1. It has fused rings labeled ABCD (present in all steroids). 2. It has 2 angular methyl groups at position 10 and 13; the methyl carbons are C19 and C Side chain is bonded to carbon 17. There are 2 configurations, cis and trans A/B junction for all naturally occurring steroids could be cis or trans while B/C acid C/D are always trans In natural cholesterol, C19 is up (above the plane of the ring).

45 Functions of cholesterol 1. Component of cell membranes 2. Synthesis of bile acids 3. Antheroclerosis 4. Synthesis of sex hormones 5. Vit D synthesis

46 Biosynthesis of cholesterol Cholesterol is synthesized from isoprene units origin from isopentenyl pyrophosphate. Squalene oxidocyclase catalyzes a series of electron shifts, initiated by protonation of the epoxide, resulting in cyclization Structural studies of a related bacterial enzyme have confirmed that the substrate binds at the active site in a conformation that permits cyclization with only modest changes in position as the reaction proceeds

47 The product of the cyclization reaction is the sterol lanosterol Conversion of lanosterol to cholesterol involves 19 reactions, catalyzed by enzymes associated with endoplasmic reticulum membranes Additional modifications yield the various steroid hormones or vitamin D

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49 Many of the reactions involved in converting lanosterol to cholesterol and other steroids are catalyzed by members of the cytochrome P450 enzyme superfamily The human genome encodes 57 members of the cytochrome P450 superfamily, with tissue-specific expression and intracellular localization highly regulated. Some P450 enzymes are localized in mitochondria. Others are associated with endoplasmic reticulum membranes Cytochrome P450 enzymes catalyze various oxidative reactions

50 Many are mixed function oxidations (mono-oxygenations), that require O2 as well as a reductant such as NADPH One oxygen atom is incorporated into a substrate and the other oxygen atom is reduced to water An example is hydroxylation of a steroid, as in the endoplasmic reticulum electron transfer pathway depicted at right, NADPH transfers 2 electrons to cytochrome P450 via a reductase that has FAD and FMN prosthetic groups.

51 P450 substrates include steroids, polyunsaturated fatty acids, eicosanoids, retinoids, and various non-polar xenobiotics (drugs and other foreign compounds) Some P450 enzymes have broad substrate specificity. Mechanisms for detoxification of non-polar compounds include reactions such as hydroxylations that increase polarity, so that the products of these reactions can be excreted by the kidneys

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53 Chemistry of steroids 1. Steroids undergo reactions characteristic of the functional groups presents 2. Reactions are greatly influenced by stereochemistry and steric factors-the fused ring presents a rigid structure. 3. There are 2 angular methyls on the B face. Therefore many reagents preferentially attack at the relatively unhindered and face especially when the reactions take place near the angular methyls

54 Reactions a) Hydrogenation occurs across the double bond. It is possible for the Hs to attack at the α-face because the angular methyl in on the B face H 2 /Pt HO b) Esterification HO H H RCOOH HO HO O HO OH Nu

55 Bile acids These are isolated from the bile of higher animals. They occur as salts or amides of either glycine or taurine This makes the molecule partly polar and partly non polar. Bile acids are synthesized from cholesterol Bile acids resemble cholesterol, but have more polar constituents Like the fatty acids, which have polar and non-polar portions that allow them to act like detergents, the bile acids also act like detergents

56 They are stored in the bile in the gall bladder and act to solubilize dietary lipids during digestion Glycocholate is a major bile salt Bile salts provide one way to break down cholesterol in the body and, as noted in the previous paragraph, blocking their normal reabsorption in the intestine requires the body to break more cholesterol down for their synthesis

57 Their functions include 1. They are emulsifying agent in the gut, acids indigestion of liquids 2. They provide suitable ph for action of lipases in the small intestine 3. They are metabolites of cholesterol 4. They promote transport of liquids through aqueous medium (blood and body fluids)

58 Positions of OHs are 3, 6, 7, 11, 12 and 23. In all natural bile acids have 3 OHs.

59 Vitamin D Synthesis Vitamin D is derived from a cholesterol derivative, 7- dehydrocholesterol, in a reaction that is initiated by ultraviolet light and which splits one of the rings The active form, calcitrol, is formed from Vitamin D3 by hydroxylation reactions in the liver and kidney Vitamin D deficiency causes a syndrome known as rickets, which is characterized by inadequate calcification of cartilage and bone Vitamin D deficiency in children was signficant in 17th century England. Today, many foods, including milk are fortified with vitamin D

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61 HORMONES Hormones are substances or chemical messengers produced by endocrine glands that elicit specific effects in target tissues or organs They are required in minute quantities and are short-lived. E.g. insulin, glucagon, sex hormones, adrenocortical hormones, gastrointestinal hormones etc. An example of a plant hormone is ethylene (ethane). It regulates plant growth and fruit ripening and controls sex of flowers of certain plants 3-indole acetic acid in a naturally occurring hormone in plants that catalyzes fruit development, stem elongation, ethylene stimulation and synthesis, begins root formation in cuttings, decreases fruit loss and spoilage when the fruit drops from the tree

62 Steroid hormones The most important steroid hormones in vertebrates are listed below

63 Calcitriol (vitamin D hormone) is also included in this group, although it has a modified steroid structure. The most important steroid hormone in invertebrates is ecdysone. Progesterone is a female sexual steroid belonging to the progestin (gestagen) family. It is synthesized in the corpus luteum of the ovaries The blood level of progesterone varies with the menstrual cycle. The hormone prepares the uterus for a possible pregnancy Following fertilization, the placenta also starts to synthesize progesterone in order to maintain the pregnant state.

64 The development of the mammary glands is also stimulated by progesterone. Estradiol is the most important of the estrogens. Like progesterone, it is synthesized by the ovaries and, during pregnancy, by the placenta as well Estradiol controls the menstrual cycle. It promotes proliferation of the uterinemucosa, and is also responsible for the development of the female secondary sexual characteristics (breast, fat distribution, etc.).

65 Testosterone is the most important of the male sexual steroids (androgens) It is synthesized in the Leydig intersitial cells of the testes, and controls the development and functioning of the male gonads It also determines secondary sexual characteristics in men (muscles, hair, etc.).

66 Cortisol, the most important glucocorticoid, is synthesized by the adrenal cortex It is involved in regulating protein and carbohydrate metabolism by promoting protein degradation and the conversion of amino acids into glucose As a result, the blood glucose level rises. Synthetic glucocorticoids (e. g., dexamethasone) are used in drugs due to their anti-inflammatory and immunosuppressant effects. Aldosterone, a mineralocorticoid, is also synthesized in the adrenal gland. In the kidneys, it promotes Na + resorption by inducing Na + /K + ATPase and Na + channels.

67 At the same time, it leads to increased K + excretion. In this way, aldosterone indirectly increases blood pressure. Calcitriol is a derivative of vitamin D. On exposure to ultraviolet light, a precursor of the hormone can also arise in the skin Calcitriol itself is synthesized in the kidneys. Calcitriol promotes the resorption of calcium in the intestine and increases the Ca 2+ level in the blood.

68 Iodothyronines The thyroid hormone thyroxine (tetraiodothyronine, T4) and its active form triiodothyronine (T3) are derived from the amino acid tyrosine The iodine atoms at positions 3 and 5 of the two phenol rings are characteristic of them Post-translational synthesis of thyroxine takes place in the thyroid gland from tyrosine residues of the protein thyroglobulin, from which it is proteolytically cleaved before being released Iodothyronines are the only organic molecules in the animal organism that contain iodine. They increase the basal metabolic rate, partly by regulating mitochondrial ATP synthesis. In addition, they promote embryonic development.

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71 PHEROMONES Many animals and insects use chemicals to communicate with others of the same species These chemicals are called pheromones. Pheromones are substances secreted by insects or animals that trigger a reaction in a member of the same species The reaction triggered may elicit 1. Immediate behavioural effect e.g. aggregation and 2. Primer effect, not immediate, physiological effect

72 Types of Pheromones 1. Sex pheromones - this is species specific, it makes sure they find a mate 2. Aggregation pheromones - helps to exert complete attack on food or prey 3. Epideitic pheromones - helps to prevent overcrowding; chemical mediated spacing of attack. 4. Alarm pheromones - helps to raise alarm of danger to ants

73 Sex Pheromones characteristics 1. Airborne 2. Contact They are perceived by antennae which have sense cells. Pheromones act on the sensory nerve cells at the mouth parts or antennae

74 Applications of pheromones 1. Monitoring population. Sex pheromone can be used to lure and trap pests or insects. We can set up a trap and put in it sex pheromone of a particular insect. 2. Pheromones can be used to cause disruption of mating 3. Pheromones can be combined with conventional insecticides to control insect infestation of agricultural fields.