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Biosynthesis of Alkaloids Alan C. Spivey

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Biosynthesis of Natural Products Biosynthesis of Alkaloids Alan C. Spivey [email protected] Oct 2019 Format & Scope of Lecture pyridine N pyrrolidine • What are alkaloids? N – definitions, 1° metabolism → a-amino acids (Lys, Orn) H • the citric acid cycle – oxaloacetate & a-ketoglutarate • pyridoxal – transamination, racemisation & decarboxylation piperidine N • Phenylalanine & tyrosine derived alkaloids H – benzylisoquinolines – e.g. opium, aporphine & erythrina alkaloids HN • Tryptophan derived alkaloids tropane – mixed tryptophan/mevalonate (isoprenoid) alkaloids: • secologanin derived: vinca-, strychnos- & quinine alkaloids etc. pyrrolizidine • Non-ribosomal peptides & derivatives N – penicillins & cephalosporins quinolizidine N indolizidine N Alkaloids • Definitions: – originally – ‘a natural product that could be extracted out of alkaline but not acidic water’ (i.e. containing a basic amine function that protonated in acid) – more generally - ‘any non-peptidic & non-nucleotide nitrogenous secondary metabolite’ H N O OH HO H OH N Me H N N coniine nicotine O N H H CO2H retronecine N clavulanic acid H N H ALKALOIDS H HO N H HO sparteine MeO N HO2C O N NMe quinine H NMe H H H H HO N morphine H H O H O NH strychnine lysergic acid a-Amino Acids used to make Alkaloids tryptophan phenylalanine tyrosine lysine ornithine Primary Metabolism - Overview For interesting animations’ of e.g. photosynthesis see: http://www.johnkyrk.com/index.html The Citric Acid Cycle • The citric acid (Krebs) cycle is a major catabolic pathway of 1° metabolism that provides two key building blocks for aliphatic amino acid biosynthesis - oxaloacetate & a-ketoglutarate: CO2 NADH CO 2 SCoA O CoA O OVERAL STOICHIOMETRY pyruvate acetyl coenzyme A NAD CO2 1x O CO2 CO O CO H2O 2 2 HO 'acetate' NADH CO O 2 CO2 + HO CO2 CO2 CO2 CO2 CO2 H2O NAD oxaloacetate citrate cis-aconitate 1x O2 OH CO2 CO2 CO2 malate isocitrate aliphatic amino acids NAD NADH H2O O CO CO 2 CO2 2 GTP NADH FADH2 O CO2 CO2 O SCoA CO2 2x CO2 CO2 CoA CO2 CO2 fumarate FAD oxalosuccinate + Pi + GDP CoA CO CO2 CO2 2 succinate succinyl-SCoA NAD a-ketoglutarate 12x ATP energy! THE CITRIC ACID CYCLE The Biosynthesis of Lysine & Ornithine • Lysine & ornithine - the two most significant, non-aromatic a-amino acid precursors to alkaloids: – NB. lysine (Lys) is proteinogenic whereas ornithine (Orn) is not – phenylalanine (Phe), tyrosine (Tyr) & tryptophan (Trp) from shikimate are the other important precursors – biosynthesis is via reductive amination of the appropriate a-ketoacid mediated by pyridoxal-5’-phosphate (PLP) O reductive NH2 NH3 O amination OH O R R R O O O ketoacid amino acid NH3 O H3N NH3 O O O O O CHO tightly lysine (Lys) pyridoxamine P O P O pyridoxal O O bound to O O [50 ATP equivs] phosphate enzyme phosphate N Me N Me H H NH3 H3N O O NH3 O O O O O ornithine (Orn) O O oxidative O O [<44 ATP equivs (=Arg)] a-ketoglutarate deamination glutamic acid (Glu) citric acid cycle OVERALL: TRANSAMINATION PLP Chemistry – Transamination & Racemisation • Transamination – LHS → RHS (reductive amination); RHS → LHS (oxidative deamination): Enz-NH3 Enz-NH2 H Enz-NH2 R CO R CO R CO2 2 2 Enz NH3 R CO2 H N N N N OP OP H OP H OP H OP H O imine O O O O exchange O N N N N N H H2O H H H H H R CO2 pyridoxamine imine pyridoxal phosphate bound as imine phosphate formation transaminase NH3 • Racemisation/inversion of configuration: H Enz-NH2 Enz-NH H 3 Enz-NH2 Enz R CO2 H R CO2 R CO2 R CO 2 imine N Enz NH exchange OP H 3 N N N OP H OP H N O OP H OP H O O O O imine H N exchange R CO2 H N N N N H H H H pyridoxal phosphate NH3 bound as imine pyridoxal phosphate racemase bound as imine PLP Chemistry – Decarboxylation • Decarboxylation: • Decarboxylation of lysine & ornithine: PLP dependant PLP decarboxylase piperidine alkaloids O HO2C NH2 NH2 H2N NH2 RHN NH2 RHN N CO2 R lysine cadaverine iminium salt PLP dependant decarboxylase PLP pyrrolidine alkaloids HO C NH NH H N NH RHN NH O N 2 2 2 2 2 2 RHN R CO ornithine 2 putrescine iminium salt Control of PLP Activity – Stereoelectronics • How does an enzyme control whether the PLP co-factor effects racemisation or decarboxylation? – i.e. which bond will be cleaved? Phenyalanine & Tyrosine Derived Alkaloids • Alkaloids (generally) containing an ArC2N subunit (± ArC2/ArC1): – Skeleta built up by reductive amination, decarboxylation, oxidation (e.g. phenolic coupling, hydroxylation) – Major classes: – monocyclic alkaloids – phenethylamines (e.g. mescaline)] – Benzylisoquinolines – opium alkaloids (e.g. papaverine, morphine) – aporphine alkaloids – erythrina alkaloids – amaryllidaceae alkaloids – e.g. lycorine, galanthamine Benzylisoquinoline Opium Alkaloids Benzylisoquinoline Alkaloids papaverine morphine Benzylisoquinoline Alkaloids – Ring Formation • Benzylisoquinoline alkaloids constitute an extremely large and varied group of alkaloids – many, particularly the opium alkaloids (e.g. papaverine, morphine) are biosynthesised from two molecules of tyrosine via nor-coclaurine (and then nor-laudanosoline). – Mechanism of Pictet Spengler reaction: Benzylisoquinoline Alkaloids - Papaverine • Papaverine: analgesic contsituent of the opium poppy (Papaver somniferum): – biosynthesis: – NB. The prefix nor means without a methyl group. Coclaurine, reticuline and laudanosine are the N-methyl compounds Oxidative Phenolic Coupling – Morphine • Morphine: analgesic & sedative contsituent of the opium poppy (Papaver somniferum): – biosynthesis: o-/p- oxidative phenolic coupling of reticuline: – Morphine acts by activating the opiate receptors in the brain (IC50 3 nM) – The natural ligands for these receptors are peptides: e.g. Leu-enkephalin (Tyr–Gly–Gly–Phe–Leu) (IC50 12 nM) Tryptophan Derived Alkaloids • Alkaloids containing an indole subunit: – Skeleta built up by reductive amination, decarboxylation & hydroxylation) – Major classes: – simple derivatives (e.g. serotonin, bufotenine) – mixed Trp/mevalonate alkaloids e.g. • ergot [DMAPP derived] (e.g. ergoline, lysergic acid) • vinca [secologanin derived] • yohombine [secologanin derived] • strychnos [secologanin derived] • quinine [secologanin derived] Dimeric Indole Alkaloids – Vinca extracts Dimeric Indole Alkaloids vinblastine (R = Me) vincristine (R = CHO) Potent anti tumour alkaloids used in cancer chemotherapy Tryptamine + Secolaganin → Strictosidine • Most alkaloids of mixed Tryptophan/mevalonate biogenesis (>1200) are derived from strictosidine: – Strictosidine is derived from the condensation of tryptamine with the iridoid C10 monoterpene secologanin: OPP O see isoprenoid lectures HO H OGlu CO2 HO H O tryptophan MeO2C enzymatic mevalonate (x2) geranyl pyrophosphate secologanin Pictet-Spengler N NH reaction H H H OGlu CO H isoprene 2 O H O MeO C PLP 2 2 NH3 NH2 strictosidine N N H CO2 H tryptophan tryptamine – Mechanism of Pictet-Spengler reaction: • via spirocyclic intermediate then Wagner-Meerwein 1,2-alkyl shift: NH NH NH NH 2 N N NH N N O Wagner-Meerwein H H H H H H H H H H N H OGlu OGlu 1,2-alkyl shift OGlu OGlu H + OGlu H H H H H O O O O O MeO C MeO2C MeO2C MeO2C 2 MeO2C tryptamine secologanin spirocyclic intermediate strictosidine Strictosidine → Vinca, Strychnos, Quinine etc. • The diversity of alkaloids derived from strictosidine is stunning and many pathways remain to be fully elucidated: N OH N N H H H H N N H N H H N O H H H MeO2C MeO2C N OAc ajmalicine H MeO H OH Me (vinca) CO2Me MeO2C vinblastine OH (vinca) yohimbine MeO O N N H N 3 NH O N H H H H OGlu N H OH O H O aspidospermine camptothecin MeO2C (vinca) H strictosidine (isovincoside) N H N Me HO H MeO N H O N H H N O H O quinine N O H strychnine (strychnos) gelsemine (oxindole) Penicillins & Cephalosporins • Famous story of the antibiotic penicillin: – discovery by bacteriologist Alexander Fleming at St Mary’s Hospital, London (published in 1929) – isolation & development by Howard Florey & Ernst Chain at the Dunn School of Pathology Oxford University (1939-1945) • E. Lax ‘The mould in Dr Florey’s coat’ Little Brown & Co., 2004, [ISBN 0316859257] – biosynthesis extensively studied by Baldwin: • Baldwin J. Het. Chem. 1990, 27, 71 & Baldwin et al. Nature 1995, 375, 700 Primary Metabolism - Overview For interesting animations’ of e.g. photosynthesis see: http://www.johnkyrk.com/index.html.
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  • Deploying Microbial Synthesis for Halogenating and Diversifying Medicinal Alkaloid Scaffolds

    Deploying Microbial Synthesis for Halogenating and Diversifying Medicinal Alkaloid Scaffolds

    Downloaded from orbit.dtu.dk on: Sep 28, 2021 Deploying Microbial Synthesis for Halogenating and Diversifying Medicinal Alkaloid Scaffolds Bradley, Samuel Alan; Zhang, Jie; Jensen, Michael Krogh Published in: Frontiers in Bioengineering and Biotechnology Link to article, DOI: 10.3389/fbioe.2020.594126 Publication date: 2020 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Bradley, S. A., Zhang, J., & Jensen, M. K. (2020). Deploying Microbial Synthesis for Halogenating and Diversifying Medicinal Alkaloid Scaffolds. Frontiers in Bioengineering and Biotechnology, 8, [594126]. https://doi.org/10.3389/fbioe.2020.594126 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. fbioe-08-594126 October 19, 2020 Time: 19:15 # 1 REVIEW published: 23 October 2020 doi: 10.3389/fbioe.2020.594126 Deploying Microbial Synthesis for Halogenating and Diversifying Medicinal Alkaloid Scaffolds Samuel A. Bradley, Jie Zhang and Michael K.