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Home , Aporphine, Indolizidine, Secologanin, Strictosidine

Biosynthesis of Natural Products

Biosynthesis of

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 – – e.g. opium, aporphine & erythrina alkaloids HN • derived alkaloids tropane – mixed tryptophan/mevalonate (isoprenoid) alkaloids: • derived: vinca-, strychnos- & 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 H H O H O NH 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. )]

– Benzylisoquinolines – opium alkaloids (e.g. , morphine) – – erythrina alkaloids

– amaryllidaceae alkaloids – e.g. lycorine, galanthamine 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, and 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 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. , lysergic acid)

• vinca [secologanin derived]

• yohombine [secologanin derived]

• strychnos [secologanin derived]

• quinine [secologanin derived] Dimeric Indole Alkaloids – Vinca extracts

Dimeric Indole Alkaloids

(R = Me) (R = CHO)

Potent anti tumour alkaloids used in cancer chemotherapy + Secolaganin →

• Most alkaloids of mixed Tryptophan/mevalonate biogenesis (>1200) are derived from strictosidine:

– Strictosidine is derived from the condensation of tryptamine with the C10 monoterpene secologanin:

OPP O see isoprenoid lectures HO H OGlu CO2 HO H O tryptophan MeO2C enzymatic mevalonate (x2) 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 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 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) (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