The mevalonate and methylerythritol phosphate pathway The mevalonate or the methylerythritol pathway are used by nature to prepare dimethylallyl PP and isopentenyl PP as synthons for the synthesis of terpenoids. Terpenoids are derived from isoprene units which are joined in a head-to-tail or head-to-head fashion. C5 hemiterpenes C10 monoterpenes C15 sesquiterpenes C20 diterpenes C25 sesterterpenes C30 triterpenes C40 tetraterpenes Huge variety of different structures; highly complex natural products because of further skeletal rearrangements of metabolites. Furthermore, subunits of terpenes are often derived from other metabolic pathways.

Different terpene skeletons

Mevalonate pathway The mevalonate pathway was discovered in the 1950‘s, when radioactively labeled mevalonic acid was found to be incorporated in cholesterol. The mevalonate pathway was long believed to be the only mechanism to prepare isoprene; However, another pathway wa dicovered which allows the synthesis of isoprene methylerytrithol phosphate pathway (non-mevalonate pathway). Mevalonate pathway: used by eukaryotes; some bacteria; plants and animals in the cytosol and peroxisoms.

Mevalonate pathway

Mevalonate pathway is used for the synthesis of important metabolic products; most noteworthy steroids. Inhibition of enzymes responsible results in disruption of the steroid biosynthesis • Inhibition of HMG-CoA: statins (used to

lower cholesterol levels in blood)

• Inhibition of FPPS: bisphosphonates (used in the treatment of osteoporosis)

Statins

Lovostatin

Selective inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase)

Bisphosphonates

Alendronic acid Hydroxyethyliden- Diphosponat

Ibandronic acid

Bisphosphonates interrupt biosynthesis of isoprene by inhibition of FPPS; proteins are not prenylated and this results in different inctercellular communication. Osteoplasts carry a great number of prenylated proteins and undergo apoptosis regeneration of bone material

In the 1990‘s, a different pathway for the biosynthesis of isoprene was discovered. Methylerythritol phosphate pathway; non-mevalonate pathway…

Many bacteria excluselively use this pathway; several organisms (some animals; plants) are able to produce isoprene either way (different compartments in cells)

Methylerythritol phosphate pathway

Comparison of mevalonate (a) and methylerythritol (b) pathway

Labeling experiments (14C labeled glucose) gave interesting results which could not be explained with the mevalonate pathway. These discrepancies led o the discovery of the methylerythritol phosphate pathway. The methylerythritol pathway might even be the more commonly used route and is predominant in most organisms.

Elucidation of the biosynthetic pathway for glabrol

Hemiterpenes Isoprene is a volatile compound and only few Derivatives are used in nature:

prenol tiglic acid 3-methyl-butanoic acid angelic acid

Usually, these simple derivatives are glycosylated and serve as pheromones. Esters of angelic acid (angelates) are used in traditional herbal medicine for the treatment of fever and pain. 3-Methylbutanoic acid has a unpleasant smell of sweat – however, in small conentrations, the acid and several esters have a pleasant smell and deriviatives are used in perfume industry.

Many plants (especially trees in tropical regions) produce and release huge amounts of isoprene. This release of isoprene into the atmosphere contributes to approximately 1/3 of hydrocarbon production (most important compound is methane which is mainly produced by cattle). The reason for the production and release of isoprene is not known. The gas might serve as plant pheromone or protect the tree from O3.

Monoterpenes

Monoterpenes are obtained by enzyme-catalyzed combination of dimethylallyl diphosphate (DMAPP) and isopentyl diphosphate (IPP) Formation of geranyl diphosphate

Geranyl diphosphate is the key intermediate in the biosynthesis of terpenes. Stabilization and rearrangement of the cationic intermediates explains the vast number of different structures which can be obtained from this intermediate.

Cyclization of cations

Different carbon skeletons in monoterpenes

Thujone

(+)-a-thujone (-)-a-thujone (+)-b-thujone (-)-a-thujone

Various members of the Artemisia plant family are rich in bicyclic monoterpenes (mainly thujones). Monethol odor; GABAA receptor antagonist (causes spasms and convulsions); Thujones are isolated from wormwood and are ingredients of absinthe; believed to cause halluzinations (could not be verified)

Camphor White or transparent waxy solid with strong odor. Can be isolated from several plants; most noteworthy: camphor laurel (Cinnamonum camphora). Has been known to many anient cultures – name can be traced back to a Sanskrit name. Has been used in Arabian countries and Europe since the 5th century for flavouring of dishes and drinks.

Biosynthesis

Camphor can be used to treat swellings and inflammation. Readily absorbed through skin sensation of cooling; slight anesthetic and antimicrobial properties Camphor used as chiral auxiliary in organic chemistry

Oppolzer sultam: one of the most important and often used chiral auxiliaries in synthesis. Employed in asymmetric dihydroxylations, DA-reactions, alkylations. Asymmetric Thermal Reactions with Oppolzer‘s Camphor Sultam. Kim, B. H.; Curran, D. P. Tetrahedron 1993, 49, 293.

Geraniol and related monoterpenes Important monoterpenes used in food industry and perfumes. Common ingredients in various fruits (citrus and other species)

a-Pinene and b-pinene

Common ingredient in pine-resin where it can easily be isolated from; pinenes are found in conifers and non-coniferous plants. Upon heating, pinene can be converted to myrcene which

serves as pheromone to the bark beetle.

Verbenol: attracts bark beetles;

Verbenol and verbenon are synthesized from pinene

Verbenon: serves as repellant; also present in rosemary; used in cough medicine

Menthol Obtained from peppermint / mint extracts or synthetically prepared. Waxy, crystalline substance with a melting point close to room temperature Menthol acts as local anesthetic; counterirritant properties. The terpene interacts with cold-sensitive TRPM8 receptors in the skin

Menthol (and other substances) interact with the TRPM8 receptor in the skin Cooling sensation

Capsaicin interacts with the TRPV1 receptor Sensation of heat

Different isomers of menthol

Stability of menthol can be explained by all-equatorial positiions of substituents

Biosynthesis of menthol

Synthesis of menthol Application of chiral Rh-catalyst on large scale

Pulegone

Intermediate in the biosynthesis of menthol Odor similar to peppermint and camphor. Used in flavouring agents and perfumery. Important starting material for enantioselectivetotal syntheses. Extremely popular in the 1980‘s, still used today

Irregular monoterpenes

Pyrethrins Isolated from pyrethrum flowers; important insecticidal compounds. Synthetic derivatives shown below.

Iridoids Iridoids are found in many medicinal plants and these compounds possess a wide range of biological properties: cardiovascular, analgesic, anti-inflammatory, antiviral , antitumor … Produced by plants mainly as defense agents against insects and microorganisms. Important intermediates in the biosynthesis of various indol alkaloids.

Cantharidin

Produced by some insects, escpecially members of the family of blister beetles (most noteable: Spanish fly). Poisonous compound; can be used as topical medication: removal of warts and small tattoos One of the world‘s oldest aphrodisiacs; used by ancient Romans, Marquis de Sade, many famous artists… Frequetly used as poison (Medici in 1500) Used in love charms…

Biosynthesis of cantharidin Still unknown. Several suggestions (monoterpene? Sesquiterpener? Pathway still unknown…

Sesquiterpenes Sesquiterpenes are synthesiszed from geranyl PP and isopentenyl PP. Farnesyl PP

Sesquiterpenes are commonly derived from the mevalonate pathway. In a similar fashion as monoterpenes, the great diversity of sesquiterpenes can be explained via rearrangement of carbocations.

Farnesyl PP can isomerize to E,Z-isomer or form nerolidyl PP with tertiar diphosphate moiety

A great variety of structurally complex sesquiterpenes are generated from farnesyl PP

Humulene derived sesquiterpenes

Artemisinin Important natural product with unusual strucutral features (peroxide linkage) Isolated from sweet wormwood (Artemisia annua) – originally native to temperate Asia, But naturalized throughout the world. Sweet womrwood was used in traditional Chinese medicine to treat fever and malaria – artemisinin was identified as biologically active ingredient

Artemisinin

Artemisia annum contains up to 1.4% artemisinin, but generally the yield is much lower (0.05-0.2%).. The concentration of artemisinic acid is much higher and the anti-malaria drug can be semisynthetically prepared. Genes responsible for the encoding the enzymes required for the biosynthesis of artemmisinic acid have been cloned in S. cerevisiae. Today, artmeinisin is prepared semisynthetically from biotechnologically derived artemisinic acid.

Synthesis of artemisinin from artemisinic acid

Malaria

• Anopheles mosquito – Plasmodium falciparum

• Course of disease – Incubation periode 10-30 days – Symptoms

• Headache • Lassitude • Vague pain in bones and joints • Fever (~40°C) • Cramps

– Cyclic pattern (paroxysm) of chill-fever -relative normalcy in an

interval of 36 hours – Coma (P. faciparum breached the blood-brain-barrier) – Death (if untreated)

http://www.youtube.com/watch?feature=player_detailpage&v=859YQcqYPko

Plasmodium life cycle

Malaria – The Plasmodial Life Circle

Malaria

• Epidemiology

– estimated 200 million global cases

– mortality of more than one million people/year

Historically important pesticide: DDT (dichlorodiphenyltrichloroethane)

First synthesized in 1874 but insecticidal properties not discovered until 1939. Most importatn insecticide during World War II. The compound is extremely stable and shows low biodegradability; accumulates in fatty tissue; might causes birth defects in humans; extremely toxic to certain animals

Gossypol

Phenolic natural product derived from cotton plants (genus Gossypium). Yellow pigment. Gossypol was responsible for low fertility in male plantation workers; has been evaluated as male oral contraceptive; however, some patients suffered permanent infertility. Binds to calmodulin (Ca-binding messenger proteins); antimalarial and anti-HIV properties

Protoilludanes Protoilludanes can be isolated from various mushrooms and fungi. Some protoilludanes possess moderate antibacterial and insecticidal properties – compounds act as defense mechanism for these mushrooms.

Hirsutane skeleton – hirsutene

Member of the triquinane family of natural products. Hirsutene and derivatives are isolated from various fungi (hirsutic acid: Sternum hirsutum) Hirsutene has served as playground for organic chemists. More than 25 syntheses of hirsutene have been reported so far. Pirrung, M. C.; Morehead, A. T.; Young, B. T. in The Total Synthesis of Natural Products, Wiley New York, 2000, Vol. 112, pp. 275-357.

Banwell’s synthesis of hirsutene

List’s synthesis of hirsutene

Diterpenes

Some important members of the diterpene family

Taxol

Extremely important anticancer drug (lung, ovarian, breast cancer) isolated from the bark of the Pacific yew tree (Taxus brevifolia); produced by endophytic fungi in the bark of the tree). Stabilizes mictotubuli and prevents cell division. Today prepared by semisynthesis

Biosynthesis of taxol

Steviol Steviol was first isolated in 1931 from Stevia rebaudiana, endemic to tropical and subtropical regions in America. The plant has been cultivated for ist sweet leaves. Steviol extracts up to 300 times sweeter than sugar and is now used as a substitute for sugar (low calories, can be consumed by patients with diabetes mellitus. The incorporation to the markest took several years and is still controversial (sugar industry).

Ginkgo biloba Ancient plant; ony survivor of the Ginkgoaceae plant family; All other members are extinct. Ginkgo extracts are used to treat cerebral vascular disease. Terpenoids present in ginkgo extracts improve peripheral and cerebrovascular circulation. Thus, ginkgo extracts are also used to treat symptoms such as vertigo, tinnitus and hearing loss (these symptoms can arise from blood circulation problems). Ginkgo extracts also act as radical scavengers (high flavonoid content). However, some of these phenols are allergenic and might induce contact dermatitis.

Forskolin and related diterpenes Labdane diterpene; isolated from the Indian Coleus plant (Coleus forskolii, also known as Plectranthus barbatus) The plant has a long history in traditional folk medicine. Forskolin raises the level of cyclic AMP, an important second messenger, responsible for signal transduction as some hormones (glucagon, adrenalin) cannot pass through cell membranes. • Inhibition of colon cancer cell growth • Potential as vasodilator (widening of blood vessels) • Increases rsistance of skin towards UV-irradiation

(topical application in tanning solution) • Treatment of urinary tract infections

Cascade cyclization to form bicyclic and poylcyclic natural products. Trans-decalin systems are favored because of low sterical hindrance of substituents.

General Information on Euphorbiaceae

• One of the largest family of flowering plants with approximately 2000 species. • Worldwide distribution with huge variation of the different members • Economically important members: Hevea brasiliensis (natural rubber), Croton tiglium (source of croton oil) and Manihot esculenta (among top ten food crops). • Plants for gardening, for example Euphorbia pulcherrima (poinsettia) • Some members of this family have irritant lattices or seed oil with a high content of terpene-based natural products

lathyrane daphnane rhamnopholane crotopholane

jatrophatrione-type presegetane myrsinane euphactine

Structural Diversity in Euphorbiaceae Natural Products: Tricyclic Diterpenes

tigliane ingenane jatropholane euphoractine-type segetane

cyclomyrsinane euphoreppine-type paraliane euphoractine-type

Structural Diversity in Euphorbiaceae Natural Products: Tetracyclic Diterpenes

Euphorbiaceae as Source of Structurally Challenging, Biologically Active Natural Products

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Multidrug Resistance Effect (MDR) 1

• Chemotherapy can fail due to the development of tumor cell resistance to multiple drugs.

• The most important mechanism of MDR is the overexpression of active drug efflux pumps in the

membranes of cancer cells.

Multidrug Resistance Effect (MDR) 2

• The efflux pump proteins belong to the ATP-Binding Cassette (ABC) superfamily of proteins.

• The members of this family mediate a variety of transport processes in healthy procaryotic and eucaryotic cells.

Transmembrane efflux pump A: X-ray crystal structure of a bacterial multidrug efflux pump B: cryoelectron microscope structure of mammalian Pgp

Multidrug Resistance Effect (MDR) 3

• 15 ABC proteins have been identified to export chemotherapeutics in in vitro experiments.

• Only 3 transporters have been implicated as major contributors to MDR in cancer (Pgp, MRP1, ABCG2).

• A variety of compounds were identified as inhibitors that can reverse MDR. As a result, cytotoxicity is restored and the cell is killed.

• Several structurally diverse diterpenes of the jatrophane skeleton, among those Pl-3 and Pl-4, were found to be highly active MDR modulators.

Biologically most active Euphorbiaceae diterpenes

Sesterterpenes

Triterpenes

Triterpenes are not accessed from geranylfarnesyl PP but obtained from two molecules of farnesyl PP – head-to-tail coupling. Squalene serves as starting material for the biosynthesis of steroids and related natural products

Lanosterol is converted into cholesterol and steroids.

Conversion of lanosterol to cholesterol proceeds via loss of three carbons

Cholesterol

Cholesterol serves multiple purposes and is a key intermediate in the synthesis of steroids • Substrate for the synthesis of vitamin D • Biosynthesis of mammalian sex hormones • Production of bile – responsible for the digestion of lipids

Cholesterol is part of the cell membrane; responsible for membran permeability and fluidity

Cholesterol is produced in all cells, but 20-25% are produced by liver cells; transported through the body with special proteins in the blood. Only minor amounts of chlesterol is obtained from dietry sources (main sources in food: cheese, egg, beef, poultry, fish) An average 70 kg person produces approximately 1 g of cholesterol per day; total cholesterol level: 35g Cholesterol is insoluble in water (blood): Different transporter proteins are used HDL: high density lipoprotein LDL: low density lipoprotein Huge proteins with up to 1500 molecules of cholesterol bound; mass of 3 million Dalton Distinct correlation of blood cholesterol level and cardiovascular problems.

Bile

Cholic acid Chendodeoxycholic acid

Taurocholic acid

Glycholic acid

Deoxycholic acid Lithocholic acid

Bile is produced by liver cells and used for the digestion of lipids; emulsifies the fats in food; The higher surface than helps to increase the efficiency of pancreatic enzymes (lipases)

Liver cells with droplets of bile

Gallstones are mainly insoluble derivatives of cholesterol and cholesterol which is required for the biosynthesis of bile

Vitamin D Group of fat-soluble steroids which play a major role in the intestinal absorption of calcium and phosphate. Vitamin D differ from other vitamins as humans can produce it – we do not solely depend on dietry sources. If exposed to sunlight, vitamin D is prepared from cholesterol and related steroids in the skin. Additional vitamin D is obtained from meat, fish, eggs… usually added to milk and milk products to reduce risk of deficiency. Deficiencies of vitamin D results in cardiovascular problems, multiple sclerosis

Vitamin D

Cardioactive glycosids

Digitalis purpurea Bufo toads

Several steroids are able to strengthen a weakened heart. The number of heartbeats is reduced and the force of the heart muscle is increased. Cardioactive glycosides are mainly produced by plants – only a small number of animals have been identified which produce these compounds.

Cardioactive steroids are characterized by a cis-fused AB-decalin system

Biosynthesis of cardioactive steroids starts with cholesterol

The biological activity of cardiactive steroids (glycosids) strongly depends on the structure of the aglycone and the sugars attached to the steroid. Isolation of cardioactive drugs from plant sources is a highly delicate process.

Levaes of the plants (mainly Digitalis purpurea) have to be dried at 60 °C immediately after the harvest in order to prevent the cleavage of the glycosidic bond.

Concentration and glycosylation pattern varies in differernt batches. Every extract needs to be tested (usually on guinea pig)

Corticosteroids

Corticosteroids Biosynthesis takes place in the adrenal cortex (outer part of adrenal glands) Two main activity groups: glucocorticoids: responsible for the synthesis of sugars from proteins and

glycogenformation / deposition in the liver

mineralcorticoids: responsible for electrolyte balance (retention of Na+, Cl-, K+)

Biosynthesis starts with cholesterol or progesterone and involves leavage of the side shain (compare to biosynthesis of cardioactive steroids)

Biosynthesis starts from cholesterol

The medicinal use of corticosteroids started after the observation that cortisone reduces symptoms of rheumatois arthritis. Initially, cortisone was isolated from adrenal glands of cattle and later prepared via a 30 step synthesis from deoxycholic acid (see chapter on bile) Cortisone is not the acive compound, the steroid is reduced in the liver to hydrocortisone which is the active agent. Cortisone has serious side effects if administered over a prolonged time or in larger amounts. Several corticosteroids have been developed with reduced side effects, a variety of different compounds is in use today. Hydrocortisone reduces inflammation and is responsible for immunosuppression.

Semisynthesis of corticosteroids

Sex hormones Responsible for the development of sexual characteristics, pregnancy…

Female sex hormones: Progestogens: responsible for preparing theuterus for pregnancy; most important steroid: progesterone

Oestrogens: mainly produced during pregnancy; responsible for menstrual cycle

Sex hormones Male sex hormones: Testosteron: Development and maintenane of male sex characteristics; stimulates growth of bones and muscles. (low levels of testosterone are also prduced by females)

Tetraterpenes Only one group of natural products is knows: carotenoids

Vitamin A Group of fat soluble vitamins; only found in animals (mainly eggs, dairy products, liver, kidneys). Precursors (provitamins) are present in many plants, especially green vegetables. Provitamins are converted to vitamin A in the liver Vitamin A is important vision (night vision), embryonic development, cell differentiation, growth

Esterification of the vitamin creates a better leaving group in the isomerization reaction

Higher terpenoids Natural ruber and Gutta percha are the most important naturally occuring isoprenoid polymers

Natural rubber

Natural rubber was used by indigenous people; natural rubber polymerizes and can be used for tools… In 1839, Goodyear invented the vulcanization process and initiated polymer chemistry. Cross-linked polymers showed better properties and rubber could be used for tires…