Heroin (diacetyl morphine or diamorphine) a semisynthetic derivative of morphine. Me Me N. Strychnine Toxin from Strychnos nux-vomica (tree)

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1 Alkaloids

2 Alkaloids Morphine Papaver somniferum (Plant) eroin (diacetyl morphine or diamorphine) a semisynthetic derivative of morphine Codeine (methylmorphine) lysergic acid ergot fungi Caffeine Isolated from plants - first 1819 Stimulant Alkaloid Coniine eurotoxin from Conium maculatum (Plant) Strychnine Toxin from Strychnos nux-vomica (tree) C Bz Ac Aconitine Toxin from aconite species (plant) Cl icotine Stimulant from the icotiana (ightshade) genus (Plants) Epibatidine Pain killer (200 x more potent than morphine) Epipedobates tricolor (Frog) Cocaine Erythroxylum coca (Plant) Alkaloid

3 Cocaine The Coca plant (Erythroxylum coca) grows in E parts of South America. Used for at least 3000 years by natives. Introduced into Europe during 16th century. Popular in drinks, including coca-cola. Albert iemann (Gottingen) isolated pure cocaine in First synthesised and its structure elucidated in 1898 (Richard Willstater, Munich, obel prize 1915). Pure cocaine used as an anaesthetic - e.g. Koller 1884 experimented on his own eye. Freud wrote about the euphoric effects of ingestion of pure cocaine at the same time. Tropinone Scopolamine yoscyamine Cocaine Efforts to Understand Cocaine Biosynthesis led to important developments in methodology for the wider investigation of secondary metabolism in plants and microorganisms Erythroxylum coca Atropine Cocaineisatropanealkaloid-namedafterthe 7 tropinone nucleus. ther highly bioactive compounds in this class include scopolamine and hyoscyamine species e.g. the nightshade family of plants). Atropine is an important pharmaceutical - it is a racemic mixture of hyoscyamine enantiomers.

4 The concepts of Labelling and Dilution endogenous unlabelled precursor endogenous unlabelled precursor in excess much is diverted much does not penetrate the cell endogenous labelled precursor otes Label is inevitably diluted - it is usual for only a small proportion to be incorporated. Labelled Precursors Isotope natural abundances % 3 - trace * much is diverted CELL Biosynthesis A very good level of labelling would be 10% - i.e. 10% of the isolated compound contains a labelled fragment. This could easily be detected by 13 C MR (where 1.1% of endogenous molecules are labelled. 13 C - 1.1% 14 C % * 1% incorporation could be detected as an approx doubling of a 13 C peak % % 33 S % 0.1% incorporation would be hard to observe by 13 C MR - but easy by 2 MR ( 2 natural abundance is 0.015%) 34S % 35 S - trace * * radioisotope Isolated material ready for analysis - - here 1/12 = 8.3% incorporation

5 Experiment by Edward Leete (Chem Commun, 1980, p1170) Radio-labelling x 10 8 dpm painted on leaves 28 days extract leaves & purify 5 1 = 14 C Erythroxylum coca labelled ornithine 283mg, 2.4 x 10 5 dpm / mmol aq Cl, Δ PhMgBr 1/5 1/5 Ph Ph 2.4 x 10 5 dpm / mmol 1/5 1/5 2.5 x 10 5 dpm / mmol Cr 3 2 S ecgonidine 2.4 x 10 5 dpm / mmol benzoate cold KMn 4 1/5 Ph 1.2 x 10 5 dpm / mmol This experiment proves that either or both of the bridgehead carbons 1 and 5 derive from C-5 of ornithine. Experiment required extensive small scale degradation to determine the location of the label. ard work!

6 ext - Formulate a Biosynthetic ypothesis: X symmetrical intermediate 2 X X X + X Cocaine

7 Stable Isotope labelling Et Et = 13 C painted on leaves Erythroxylum coca 28 days extract leaves & purify PPM PPM C spectrum of unlabelled cocaine 13 C spectrum of cocaine from experiment Much better experiment - less work and 13 C MR precisely locates position of label. Enhancement of C-5 signal supports intermediacy of the -methyl pyrrolinium species. atural abundance of 13 C is 1.1% - detected a 0.25% incorporation

8 Dual label experiment painted on leaves 28 days extract leaves & purify = 13 C Erythroxylum coca B C Expansion of 13 C MR spectrum for C-2 A = doublet due to coupling to C-9 (incorporated 13 C) B = C-2 of cocaine (natural abundance) C = C-2 of trans-cinnamoylcocaine (minor impurity) D = C-2 of cis-cinnamoylcocaine (minor impurity) E = doublet due to coupling to C-9 (incorporated 13 C) A J = 60 z D E In an unlabelled 13 C spectrum no 13 C- 13 C coupling is observed because the chance of observing two contiguous 13 C labels is x = Dual 13 C labelling enhances sensitivity - no enhancement could be observed at C-2 due to ca 0.08% incorporation compared to 1.1% natural abundance. Coupling to C-9 proves intact unit has been incorporated. Coupling is only observed because the small proportion of molecules with 13 C at C-2 also have 13 C at C-9.

9 Cell Free extract and purified enzymes... roots homogenized Datura innoxia and centrifuged to make a cell-free extract (CFE) Tropinone 2h, 20 C AD(P) Tropine + ψ-tropine Two enzymes were eventually purified from the CFE - one which synthesises Topine and a second which synthesises ψ-tropine. The ψ-tropine reductase has been crystallised and studied. See Structure of tropinone reductase-ii complexed with ADP + and pseudotropine at 1.9 A resolution: implication for stereospecific substrate binding and catalysis. Yamashita, A., Kato,., Wakatsuki, S., Tomizaki, T., akatsu, T., akajima, K., ashimoto, T., Yamada, Y., da, J., Biochemistry 1999, 38, Crystal structure of tropinone reductase II bound to ADP and ψ-tropine.

10 Experiment by Edward Leete and Leo Vining (Tet. Lett, 1975, 47, p4103) More isotopic labelling experiments - microbial cultures Beauveria bassiana An isect pathogenic fungus... Can be grown in the lab on agar plates ca 4% incorporation of labelled phenylalanine - note enhanced doublets from now adjacent labels 2 = 13 C Phenylalanine feed to cultures 7 days and in liquid media as 'shake cultures' which are characteristically orange 5 4 Tenellin - yellow + osporein - red

11 Alkaloids The opium poppy, exuding latex which contains the opiates Papaver somniferum is used as a commercial crop in numerous countries including the UK it yields around 1.5Kg pure morphine per hectare. Morphine Papaver somniferum (Plant) The latex is rich in opiates, but all parts of the plant contain valuable material. The Biosynthesis of morphine is well understood chemically and genetically. Morphine is ultimately derived from the amino acid tyrosine.

12 Alkaloids The mechanism of a Pictet-Spenglerase - Strictosidine Synthase Tryptamine 2 + Glc Secologanin (a monoterpene) Strictosidine Synthase Strictosidine Glc Vinblastine (Anti-cancer) Yohimbine (Sexual disorders) Ajmaline (Anti-arrhythmic) Camptothecin (Anti-cancer) Strychnine (Toxin) Secologanin Tyr 151 Glu 309 is 307 See 'Connor et al, J. Am. Chem. Soc., 2008, 130,

13 Alkaloids Cocaine Biosynthesis 2 2 rnithine (5 Carbons) decarboxylase (PLP) 2 2 putrescine SAM 2 Aminotransferase (PLP) Similarly 2 SCoA SCoA SCoA Pyrrolidine alkaloids e.g. Coniine Lysine (6 carbons) 2 Piperidine Alkaoids via cadaverine Toxic principal of emlock (plant). Used by Socrates to commit suicide. First alkaloid to be synthesised by Ladenburg in SCoA P SCoA SCoA Cocaine SCoA