An Overview of Biosynthesis Pathways – Inspiration for Pharmaceutical and Agrochemical Discovery Alan C. Spivey [email protected] 19th Oct 2019 Lessons in Synthesis - Azadirachtin • Azadirachtin is a potent insect anti-feedant from the Indian neem tree: – exact biogenesis unknown but certainly via steroid modification: O MeO C OAc O 2 H O OH O H O OH 12 O O C 11 O 14 OH oxidative 8 O H 7 cleavage highly hindered C-C bond HO OH AcO OH AcO OH for synthesis! H H of C ring H MeO2C O AcO H tirucallol azadirachtanin A azadirachtin (cf. lanosterol) (a limanoid = tetra-nor-triterpenoid) – Intense synhtetic efforts by the groups of Nicolaou, Watanabe, Ley and others since structural elucidation in 1987. –1st total synthesis achieved in 2007 by Ley following 22 yrs of effort – ~40 researchers and over 100 person-years of research! – 64-step synthesis – Veitch Angew. Chem. Int. Ed. 2007, 46, 7629 (DOI) & Veitch Angew. Chem. Int. Ed. 2007, 46, 7633 (DOI) – Review ‘The azadirachtin story’ see: Veitch Angew. Chem. Int. Ed. 2008, 47, 9402 (DOI) Format & Scope of Presentation • Metabolism & Biosynthesis – some definitions, 1° & 2° metabolites • Shikimate Metabolites – photosynthesis & glycolysis → shikimate formation → shikimate metabolites – Glyphosate – a non-selective herbicide • Alkaloids – acetylCoA & the citric acid cycle → -amino acids → alkaloids – Opioids – powerful pain killers • Fatty Acids and Polyketides –acetylCoA → malonylCoA → fatty acids, prostaglandins, polyketides, macrolide antibiotics – NSAIDs – anti-inflammatory’s • Isoprenoids/terpenes –acetylCoA → mevalonate → isoprenoids, terpenoids, steroids, carotenoids – Statins – cholesterol-lowering agents Metabolism and Biosynthesis Metabolism & Natural Product Diversity O Me Me HO C 2 Me N N NMe O H H H O N N H camphor caffeine HO N H MeO NH lysergic acid CO2 H2O Pi N2 N hv quinine H Me O O OH O N Me O O H CO2H N clavulanic acid O Me androstenedione N O OH nicotine patulin Metabolism • Metabolism is the term used for in vivo processes by which compounds are degraded, interconverted and synthesised: – Catabolic or degradative: primarily to release energy and provide building blocks • generally oxidative processes/sequences (glycolysis, Krebs cycle) – Anabolic or biosynthetic: primarily to create new cellular materials (1° & 2° metabolites) • generally reductive processes/sequences • These two types of process are coupled – one provides the driving force for the other: Natural product CO2 + H2O secondary metabolites 'CO2 fixation' hv complex metabolites (photosynthesis) Nutrients ATP Cell components energy energy & Growth storage release Catabolism ADP + P i O 2 Anabolism oxidative NAD(P)H reductive Energy & degredation biosynthesis Building blocks NAD(P) + H Building Blocks respiration 'nitrogen fixation' simple products (by diazatrophs, lightening, the Haber process) CO2 + H2O N2 Primary Metabolism - Overview Primary metabolism Primary metabolites Secondary metabolites CO2 +H2O 1) 'light reactions': hv -> ATP and NADPH PHOTOSYNTHESIS 2) 'dark reactions': CO2 -> sugars (Calvin cycle) HO O oligosaccharides HOHO polysaccharides HO nucleic acids (RNA, DNA) OH glycolysis glucose & other 4,5,6 & 7 carbon sugars CO2 SHIKIMATE METABOLITES PO cinnamic acid derivatives CO2 aromatic compounds + HO HO OH lignans, flavinoids PO O OH OH phosphoenol pyruvate erythrose-4-phosphate shikimate ALKALOIDS aromatic amino acids peptides penicillins CO 2 proteins cephalosporins aliphatic amino acids cyclic peptides O pyruvate tetrapyrroles (porphyrins) Citric acid cycle (Krebs cycle) saturated f atty acids FATTY ACIDS & POLYKETIDES SCoA SCoA unsaturated f atty acids prostaglandins lipids CO2 polyacetylenes O O aromatic compounds, polyphenols acetyl coenzyme A malonyl coenzyme A macrolides CoAS O HO ISOPRENOIDS terpenoids CO2 O HO steroids carotenoids acetoacetyl coenzyme A mevalonate For interesting animations’ of e.g. photosynthesis see: http://www.johnkyrk.com/index.html Shikimate Metabolites Shikimate Metabolites H NH3 H NH3 O2C CO2 CO2 NH3 NH H n O Me O (S)-tryptophan (S)-phenylalanine (ArC0) OH (ArC ) O O 3 (S)-tyrosine (ArC3) MeO scopoletin SHIKIMATE METABOLITES OH menaquinone (vitamin K2) (ArC3) (ArC ) 1 OH H H HO OH O O 3 H O O H H O O OH Me Me OH MeO OMe -tocopherol (vitamin E) OMe HO OH (ArC1) podophyllotoxin OH (ArC3) epigallocatechin (EGC) (ArC3) The Shikimate Biosynthetic Pathway - Overview • Phosphoenol pyruvate & erythrose-4-phosphate → shikimate → chorismate → prephenate: – The detailed mechanisms of these steps have been studied intensively. Most are chemically complex and interesting. For additional details see: • Mann Chemical Aspects of Biosynthesis Oxford Chemistry Primer No. 20, 1994 (key details) •Haslam Shikimic Acid – Metabolism and Metabolites Wiley, 1993 (full details and primary Lit. citations) • http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/misc/shikim.html (interesting web-site with many biosynethtic pathways) Rational Agrochemical Development – Shikimate Pathway Intervention • The shikimate biosynthetic pathway is not found in animals/humans – only in plants – selective intervention in these pathways allows development of agrochemicals with minimal human toxicity • Glyphosate (‘Roundup’) – a Monsanto agrochemical is a potent inhibitor of the conversion of 3-phosphoshikimate (3-PS) → 5-enolpyruvylshikimate-3-phosphate (5-EPS-3P) – a non-selective herbicide H NH3 CO2 CO2 CO2 CO PO 2 CO2 phosphoenol pyruvate CO (PEP) PEP 2 H (S)-tyrosine PO OH PO O + PO O CO2 OH PO OH OP OH OH Pi 3-phosphoshikimate H NH3 HO (3-PS) O 5-enolpyruvylshikimate-3-phosphate CO OH (5-EPS-3P) 2 erythrose-4-phosphate glyphosate (Roundup®) (E-4-P) inhibits this step (S)-phenylalanine PO N CO2 H2 Chorismate → Tryptophan, Tyrosine & Phenylalanine • Chorismate → anthranilate → tryptophan • Chorismate → prephenate → tyrosine & phenylalanine – NB. The enzyme chorismate mutase [EC 5.4.99.5] which mediates the conversion of chorismate to prephenate is the only known ‘Claisen rearrangementase’ Tyrosine/Phenylalanine → ArC3 Metabolites • Tyrosine & phenylalanine → cinnamate derivatives → ArC3 metabolites – coumarins, lignans (stereoselective enzymatic dimerisation) & lignins (stereorandom radical polymerisation) Primary Metabolism - Overview Primary metabolism Primary metabolites Secondary metabolites CO2 +H2O 1) 'light reactions': hv -> ATP and NADPH PHOTOSYNTHESIS 2) 'dark reactions': CO2 -> sugars (Calvin cycle) HO O oligosaccharides HOHO polysaccharides HO nucleic acids (RNA, DNA) OH glycolysis glucose & other 4,5,6 & 7 carbon sugars CO2 SHIKIMATE METABOLITES PO cinnamic acid derivatives CO2 aromatic compounds + HO HO OH lignans, flavinoids PO O OH OH phosphoenol pyruvate erythrose-4-phosphate shikimate ALKALOIDS aromatic amino acids peptides penicillins CO 2 proteins cephalosporins aliphatic amino acids cyclic peptides O pyruvate tetrapyrroles (porphyrins) Citric acid cycle (Krebs cycle) saturated f atty acids FATTY ACIDS & POLYKETIDES SCoA SCoA unsaturated f atty acids prostaglandins lipids CO2 polyacetylenes O O aromatic compounds, polyphenols acetyl coenzyme A malonyl coenzyme A macrolides CoAS O HO ISOPRENOIDS terpenoids CO2 O HO steroids carotenoids acetoacetyl coenzyme A mevalonate For interesting animations’ of e.g. photosynthesis see: http://www.johnkyrk.com/index.html Alkaloids 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 OH N Me H H N N coniine nicotine O N H H CO2H N clavulanic acid retronecine H N H ALKALOIDS H HO N H HO sparteine MeO N HO2C O N quinine H NMe NMe H H H H HO N morphine H H O H O NH strychnine lysergic acid 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 & -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 -ketoglutarate 12x ATP energy! THE CITRIC ACID CYCLE The Biosynthesis of Lysine & Ornithine • Lysine & ornithine - the two most significant, non-aromatic -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 -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 OO OO 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)] -ketoglutarate deamination glutamic acid (Glu) citric acid cycle OVERALL: TRANSAMINATION PLP Chemistry – Transamination & Racemisation • Transamination: PLP Chemistry – Decarboxylation • Decarboxylation: O O H Enz-NH3 imine R CO2 H exchange Enz H R H R H R Enz-NH Enz N NH 2 OP H 3 N N N OP H OP H N O OP H OP H O O O O