MPS 587 - Advanced Plant Biochemistry Course

Fall Semester 2011

Lecture 20

Alkaloids II

3. Tropane alkaloids 4. Nicotine alkaloids 5. Piperidine alkaloids of poison hemlock 6. Glycoalkaloids of the nightshades 7. Pyrrolizidine alkaloids

Disclaimer

As part of the alkaloid biosynthesis section of this lecture, I will cover various illicit uses and share some stories of people who have used extracts and preparations containing mind-altering alkaloids. Herewith I would like to state very clearly that this is not meant to play down the dangers associated with the illicit use of drugs, nor do I support in any way the use of such materials.

B.M. Lange (Course director)

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Tropane alkaloids

Datura stramonium

Hyoscyamus niger

Erythroxylum coca

Atropa belladonna

Source: Wikipedia

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Tropane alkaloids of the nightshades

Atropine • Extracted from deadly nightshade (Atropa belladonna) and other plants of the Solanaceae (racemic mixture of D-hyoscyamine and L-hyoscyamine). • Competitive agonist of the muscarinic acetylcholine receptors. • Blocks contraction of circular pupillary sphincter muscle (controlled by acetylcholine release). • Used as a mydriatic, to dilate the pupils. • Also used by troops as an antidote for poisoning by nerve gases that target acetylcholine receptors. • Used by Egyptian queen Cleopatra to dilate her pupils in the hope that she would appear more alluring (belladonna is the Italian word for “beautiful lady”).

Scopolamine • Obtained from henbane (Datura stramonium) and other plants of the Solanaceae. • As atropine, competitive agonist of the acetylcholine receptors; however the therapeutic range is narrower and there are more side effects; very limited use today.

Source: Wikipedia

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Hyoscyamine

• Extracted from henbane (Datura stramonium) and other plants of the Solanaceae (D-isomer is shown). • Works by inhibting the action of acetylcholine in smooth and cardiac muscle, the sinoatrial and atrioventricular nodes and the exocrine glands. • Used to provide symptomatic relief to various gastrointestinal disorders including spasms, peptic ulcers, irritable bowel syndrome, pancreatitis, colic and cystitis. • Brand names for hyoscyamine include Symax, Anaspaz, Cystospaz, Levsin, Levbid, Donnamar, NuLev, and Neoquess.

Cocaine

• Obtained from the coca plant (Erythroxylum coca).

• Stimulant of the central nervous system and an appetite suppressant, creating what has been described as a euphoric sense of happiness and increased energy. • For many decades cocaine was a key ingredient in Coca-Cola. • When the Spaniards colonized South America, they at first ignored aboriginal claims that the leaf gave them strength and energy, and declared the practice of chewing it the work of the Devil. But after discovering that these claims were true, they legalized and taxed the leaf, taking 10 percent off the value of each crop. These taxes were for a time the main source of support for the Roman Catholic Church in the region.

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Tropane alkaloids of the nightshades Source: Wikipedia

Sigmund Freud, 1884: “...exhilaration and lasting euphoria, which in no way differs from the normal euphoria of the healthy person...You perceive an increase of self-control and possess more vitality and capacity for work....In other words, you are simply normal, and it is soon hard to believe you are under the influence of any drug....Long intensive physical work is performed without any fatigue...This result is enjoyed without any of the unpleasant after-effects that follow exhilaration brought about by alcohol....Absolutely no craving for the further use of cocaine appears after the first, or even after repeated taking of the drug...”

Cocaine use is prevalent across all socioeconomic strata, including age, demographics, economic, social, political, religious, and livelihood. Cocaine in its various forms comes in second only to cannabis as the most popular illegal recreational drug in the United States, and is number one in street value sold each year (> $ 35 B/y in 2003).

Pope Leo XIII

Cocaine use Source: Wikipedia

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Biosynthesis of tropane alkaloids (Ziegler & Facchini (2008) Annual Reviews of Plant Biology 59: 735).

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Complex structure of tropinone reductase II (TR-II) with tropinone in black and NADPH in blue+grey. (b) Complex structure of tropinone reductase I (TR-I) with NADPH illustrated in blue+grey

(Stoeckigt & Panjikar (2007) Natural Products Reports 24, 1382) (Nakajima et al. (1998) Proceedings of the National Academy of Sciences in the USA 95, 4876)

Blue colour on the protein surface indicates positive charge; red colour indicates negative charge. Stereospecific fixation of the substrate tropinone is influenced in TRI by repulsion of the alkaloid nitrogen on a positively protein surface and by hydrophobic interaction. In TRII, ionic attraction of the nitrogen by a negatively charged glutamic acid side chain holds tropinone in place. The hydrogen from NADPH is transferred to yield an equatorial or axial position of the resulting hydroxyl, respectively.

Mechanism of tropinone reductases

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Trafficking in tropane alkaloid metabolism (Ziegler & Facchini (2008) Annual Reviews in Plant Biology 59, 735)

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Nicotine

• Found in the nightshade family of plants (Solanaceae), predominantly in tobacco, and in lower quantities in tomato, potato, eggplant (aubergine), and green pepper.

• Constitutes 0.3 to 5% of the tobacco plant by dry weight, with biosynthesis taking place in the roots, and accumulates in the leaves. • In lower concentrations, the substance is a stimulant and is one of the main factors responsible for the dependence-forming properties of tobacco smoking ; in higher doses, nicotine is a potent nerve poison and is included in many insecticides. • Nicotine acts on the nicotinic acetylcholine receptors. In small concentrations it increases the activity of these receptors, among other things leading to an increased flow of adrenaline, a stimulating hormone.

Nicotiana tabacum

Top Ten Tobacco Producers

(million metric tons)

China 2.51

Brazil 0.88

India 0.60

United States 0.29

Indonesia 0.14

Turkey 0.14

Greece 0.12

Argentina 0.12

Italy 0.11

Pakistan 0.08

World Total 6.38

Source: UN Food & Agriculture

Organisation (FAO) (2005)

Source: Wikipedia

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Biosynthesis of nicotine

AO, aspartate oxidase; DAO, diamine oxidase; ODC, ornithine decarboxylase; PMT, putrescine N- methyltransferase; QPT, quinolinate phospho- ribosyltransferase; QS, quinolinate synthase.

Tropane alkaloid in other nightshades

(Katoh et al. (2005) Plant Biotechnology 22, 389)

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Alteration of the wound response of wild tobacco plants (Nicotiana attenuata) by Manduca caterpillar feeding.

(A) Wounding of the leaf tissue results in a jasmonic acid burst, which is

amplified by caterpillar feeding and the application of fatty acid amino acid conjugates (FACs) from larval oral secretions to the wound.

(B) Caterpillar attack, or the application of FACs to wounds, but not wounding alone induces the production of volatiles that function as predator attractants in the plants' indirect defense.

(C) Caterpillar feeding and the application of their oral secretions to wounds cause an ethylene burst.

(D) Ethylene attenuates the wound- and jasmonic acid-induced accumulation of nicotine by suppressing the accumulation of transcripts for a key regulatory step in nicotine biosynthesis (pmt: putresine N-methyl transferase).

(E) Caterpillar attack also results in a transcriptional reconfiguration of the plant's wound-response. Addition of FACs antagonizes the wound-induced increase (W) of transcripts representing a type-I expression pattern that spreads systemically throughout the plant from the wound site. The wound-induced increase in transcripts in a type-IIa expression pattern is further amplified after application of FACs to wounds. In contrast, the genes with a type-IIb expression pattern are suppressed after wounding and further suppressed with the addition of FACs. Both type IIa and IIb patterns are found only in the leaves directly suffering the herbivore attack

Defense mechanisms in N. attenuata attacked by the nicotine-tolerant specialist M. sexta

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(Kessler & Baldwin (2002) Annual Review of Plant Biology 53, 299)

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Piperidine alkaloids of poison hemlock (Conium maculatum) Source: Wikipedia

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• Found in poison hemlock and the Yellow Pitcher Plant. It is a neurotoxin which disrupts the central nervous system. It is toxic to all classes of livestock and humans; less than 0.2g (0.007oz) is fatal to humans, with death caused by respiratory paralysis. • Coniine paralyzes muscles in the same fashion as curare. Symptoms of paralysis occur within a half hour, and death may take several hours. Tubocurarine (present in Strychnos toxifera) • Appears to have an addictive effect: goats, cows, and pigs have all shown a preference for conium-containing forage (up to the point of eventual death) if they survive initial exposure. • A useful trick to determine whether a plant is poison hemlock rather than fennel, which it resembles, is to crush some leaves and smell the result. Fennel smells like anise or liquorice, whereas the smell of poison hemlock is often described as mouse-like or musty.

Coniine Source: Wikipedia

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In ancient Greece, hemlock was used to poison condemned prisoners. The most famous victim of hemlock poisoning is probably Socrates. After being condemned to death for impiety in 399 BC, Socrates was given a potent solution of the hemlock plant. Plato described Socrates' death in the Phaedo: He walked about and, when he said his legs were heavy, lay down on his back, for such was the advice of the attendant. The man who had administered the poison laid his hands on him and after a while examined his feet and legs, then pinched his foot hard and asked if he felt it. He said "No"; then after that, his thighs; and passing upwards in this way he showed us that he was growing cold and rigid. And again he touched him and said that when it reached his heart, he would be gone. ... To this question he made no reply, but after a little while he moved; the attendant uncovered him; his eyes were fixed.

The Death of Socrates, by Jacques-Louis David (1787) Coniine

Source: Wikipedia

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Biosynthesis of coniine, a polyketide alkaloid

(Reynolds (2005) Phytochemistry 66, 1399)

5-Ketooctanal

L-Ala

gamma-Coniceine +

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Aloe ruspoliana

Structures of various piperidine alkaloids (Reynolds (2005) Phytochemistry 66, 1399)

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Exposed to light greening (glycoalkaloids!)

Kept in the dark yellowish peel

Glycoalkaloids of the nightshades

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Phytosterols

Isoprenoid pathway Biosynthesis of glycoalkaloids

Example: tomatine biosynthesis in tomato

Glycoalkaloids of potato:

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Pyrrolizidine alkaloids

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Source: Wikipedia

L-Orn

PAO

SSy

HSS

PAO (HEH-sens.)

Biosynthesis of senecionine-type pyrrolozidine alkaloids

PAO = polyamine oxidase SSy = spermidine synthase HSS = homospermidine synthase

(Hartmann & Ober (2000) Topics in Current Chemistry 209, 208)

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Evolution of homospermidine synthase function (Hartmann & Ober (2000) Plant Molecular Biology 44, 445)

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(Hartmann (2004) Planta 219, 1)

Fate of pyrrolizidine alkaloids (PAs) after ingestion

N-oxidation (senecionine N-oxidase)

• Suppression of reduction • Conjugation of reduced PA

Transfer of free base into defensive secretions

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(Hartmann & Ober (2000) Topics in Current Chemistry 209, 208)